* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Novel Fluorescent Probes Detect Different Types Of CYP450
Survey
Document related concepts
Discovery and development of neuraminidase inhibitors wikipedia , lookup
Discovery and development of integrase inhibitors wikipedia , lookup
Discovery and development of proton pump inhibitors wikipedia , lookup
Drug design wikipedia , lookup
Psychopharmacology wikipedia , lookup
Theralizumab wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Pharmaceutical industry wikipedia , lookup
Pharmacokinetics wikipedia , lookup
Neuropharmacology wikipedia , lookup
Prescription costs wikipedia , lookup
Pharmacognosy wikipedia , lookup
Discovery and development of ACE inhibitors wikipedia , lookup
Drug discovery wikipedia , lookup
Transcript
Novel Fluorescent Probes Detect Different Types Of CYP450-Drug Interactions In High Throughput Mode Gregor Zlokarnik, Laurie P. Volak, Michele M. Andrew, Chinh Tran, Thomas Cleveland, Lewis R. Makings; Chemistry Department, Aurora Biosciences Corporation, 11010 Torreyana Road, San Diego, CA 92121 0 5 C R1 15 0 20 D VividTM 3A4 Blue R2 R2 + H2CO + + 20 6 8 10 O 0 O Metabolism of VividTM 3A4 Substrates VividTM 3A4 substrates are benzyloxymethyl ethers of phenolic fluorescent dyes. Unlike their benzylic counterparts, they possess two potential sites of oxidation that can lead to release of a fluorescent metabolite. Ab initio calculations suggest that the benzylic position (marked in red) is the more likely site of metabolism (6) as hydrogen abstraction creates a more stable radical intermediate. BFC v {min } 5 -1 4 O O O 3 25 0 % Activity 400 This work was supported in part by NIH grant GM60114 O 3 2 Red Green Blue Cyan 300 200 50 100 25 0 -8 -7 -6 -5 -4 HLM 100 75 50 25 Red Green Blue Cyan Red Green Blue Cyan TM 15 -1 v {min } O O O O CN 10 5 1A 2 2A 6 2B 6 2C 8 2C 2C 9 18 2C 19 2D 6 2E 1 3A 4 3A 5 0 15 -1 v {min } VividTM 3A4 Cyan O O O O 10 5 0 Vivid Drug Licensing opportunities: Michael J. Dunn Vice President, Business Development Aurora Biosciences Corporation 11010 Torreyana Road San Diego, CA 92121 (858) 404-6769; [email protected] VividTM 3A4 substrates were treated with individually expressed human cytochrome P450 isozymes. Substrates are predominantly metabolized by CYP3A isozymes, except for coumarin-based substrates which are also metabolized by CYP2B6. (Error bars represent standard deviation). RHI 3A4 Green HLM RHI 3A4 Blue HLM RHI 3A4 Cyan HLM RHI HLM mean SD (log) mean SD (log) mean SD (log) mean SD (log) mean SD (log) mean SD (log) mean SD (log) mean 0.02 0.2 1.0 0.1 0.03 0.3 0.02 0.1 0.3 0.3 1 0.1 0.4 0.1 2 0.1 Miconazole 0.9 - 1.3 0.03 0.1 0.1 0.1 0.1 0.02 0.1 0.01 0.1 0.1 0.1 0.05 0.4 0.1 0.2 0.05 Nifedipine 10 - 22 0.4 0.1 20 0.1 0.2 0.1 2 0.1 0.8 0.1 10 0.1 1 0.1 70 0.1 Verapamil 24 - 82 0.5 0.05 80 0.01 2 0.1 10 0.2 1 0.1 30 0.04 2 0.5 100 0.04 Troleandomycin 10 - 51 1 0.1 0.2 * 0.3 0.7 0.1 5 0.05 1 0.1 7* 0.1 1 0.03 >>100 n/a Erythromycin 16 - 194 1 0.2 8* 0.1 3 0.1 100 0.2 7 0.2 50 * 0.3 2 0.1 >>300 n/a 47 1* 0.3 >100 n/a 5 0.04 30 0.04 1 0.2 40 * 0.2 2 0.03 >100 n/a 50 - 75 3 0.1 60 0.2 4 0.1 20 0.02 4 0.01 40 * 0.02 2 0.04 >400 n/a 34 10 0.2 20 0.1 5 0.04 20 0.03 4 0.05 30 0.01 4 0.1 80 0.1 Testosterone 40 - 60 (3) 0.1 >>100 n/a 5* 0.5 20 0.1 60 0.3 >100 n/a >>80 n/a >>100 n/a Progesterone 8 - 45 (4) 0.2 200 0.3 10 * 0.2 20 0.01 50 0.3 100 0.1 >>80 n/a 200 0.1 Diltiazem RHI : Recombinant Human Isozyme (baculovirus expression, insect cell microsomes); HLM : pooled Human Liver Microsomes mean : geometric mean of triplicates; SD (log) : standard deviation of geometric mean in log units; ( ) : activation; * : partial inhibition Apparent Ki values with Recombinant CYP3A4 and Human Liver Microsomes 75 50 % Activity CYP2C CYP3A4 CYP2D6 75 75 Blue Red Green 50 25 50 0 -7 -6 -5 -4 [Drug] log {M} -8 -7 -6 -5 -4 [Drug] log {M} Blue Red Green 200 25 0 0 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 [Sulfaphenazole] -8 -7 -6 -5 -4 [Warfarin] log {M} Dose-Response Curves VividTM substrates were used to assess interaction of CYP2C9 with inhibitors (A, B), and an activator (C). Assuming a two-site model for the CYP2C9 active site (or a large TM 2C9 Blue, a binding site permitting simultaneous binding of two substrates), Vivid coumarin-based substrate, is most sensitive to drug-interaction at the activator site (panel TM C). Also, lansoprazole and sulfaphenazole inhibit Vivid 2C9 Blue turnover only partially, while they fully inhibit turnover of the other substrates. The resorufin-based substrate , TM Vivid 2C9 Red is somewhat more sensitive to drug-interactions with CYP2C9 than TM Vivid 2C9 Green. XI VividTM SD (log) mean SD (log) mean SD (log) 0.09 - 1.6 0.3 * 0.1 0.01 0.1 0.6 0.1 5 - 15 3* 0.1 0.3 0.1 6 0.1 Lansoprazole 45 - 52 5* 0.1 2 0.03 10 0.2 Omeprazole 40 - 240 5* 0.1 0.4 0.03 10 0.1 Tolbutamide 96 - 371 100 0.2 > 200 n/a >>800 n/a Warfarin 0.25 - 27 (6) 0.1 10 0.1 20 0.2 Sulfaphenazole Diclofenac IX VividTM 2C9 Green 2C9 Blue 2C9 Red 2C9 Green Lit. K (µM) mean Drug mean : geometric mean of triplicates; ( ) : Kactivation; * : partial inhibition 1.5 SD (log) : standard deviation of geometric mean in log units 1.0 Apparent Ki Values with Recombinant CYP2C9 The spreadsheet lists apparent Ki values of drugs determined with VividTM 2C9 substrates and recombinant human CYP2C9 isozyme. Shown for comparison are values from the recent literature in which drug substrates and human liver microsomes were used. 2C9 Blue 0.5 2C9 Red 0.0 0 5 Lit K : Ko, J.W. et al. (1997). Drug Metab. Dispos., 25, 853; Mancy, A. et al. (1996). Biochemistry, 35, 16205 10 [substrate] {µM} Substrate Kinetics VividTM 2C9 substrates display Michaelis-Menten type kinetics. Line color indicates the color of the fluorescent metabolite generated. TM Km= 20 µM, kcat = 1.5 min-1 Km= 2 µM, kcat = 0.4 min-1 TM 2C9 Green Km= 2 µM, kcat = 3.0 min-1 Vivid 25 -8 C Blue Red Green 400 [Lansoprazole] Vivid 2C9 Blue TM Vivid 2C9 Red XII VividTM 2C9 Substrates Detect Drug Interactions 100 Assays were performed with 10nM human CYP2C9 (insect cell microsomes containing baculovirus-expressed 2C9 and NADPH-P450 reductase; Panvera, WI). 60 TM 40 20 Vivid 2C9 Green Red 0 Blue >100 Drugs ranked by Potency (Blue) Shown are the % inhibition values for >100 drugs in VividTM 2C9 assays at 10µM [drug]. VividTM 2C9 Blue detects inhibitors ( ) and activators (off scale, not shown) of CYP2C9. At the screening concentration all substrates detect the potent inhibitors of CYP2C9. CYP3A4 Assay in NanoplateTM Dose responses for Ketoconazole ( ), Verapamil ( ) and Yohimbine ( ) were measured side-by-side using the VividTM 3A4 Red substrate and recombinant human CYP3A4 in NanoplateTM (2µl) and 96-well formats (100µl). Comparable IC50 values were obtained. SD (log) 0.015 - 8 Ethinylestradiol Isozyme Selectivity of VividTM 3A4 Substrates Lit. K (µM) 3A4 Red Ketoconazole Amiodarone 1A 2 2A 6 2B 6 2C 8 2C 2C 9 18 2C 19 2D 6 2E 1 3A 4 3A 5 CF3 VI TM 96-Well Plate 100 Exemplary IC50 curves of four drugs known to interact with CYP3A4 are shown. Vivid 3A4 substrates were either used with recombinant human CYP3A4 (RHI) or with pooled human liver microsomal preparations (HLM). In general, drugs were less potent in inhibiting VividTM 3A4 probe metabolism in liver microsomes than in recombinant CYP3A4 preparations (A, B, D). Interestingly, the profound activation of VividTM 3A4 Red metabolism by testosterone in recombinant CYP3A4 occurs to a much lesser degree in human liver microsomes (C). As in the recombinant system, partial inhibition of probe metabolism (D) is found for certain drugprobe pairs in human liver microsomes, but for a different pair (B). Troleandomycin, a potent inhibitor of recombinant CYP3A4 only TM TM partially inhibits Vivid 3A4 Red metabolism. Vivid 3A4 Green turnover is partially inhibited by steroids, both in recombinant TM and liver preparations. The two coumarin-based substrates Vivid 3A4 Blue and Cyan give similar results to each other. B 100 80 NanoplateTM [Amiodarone] log {M} A 100 0 2.0 -8 -7 -6 -5 -4 -9 -8 -7 -6 -5 -4 CYP1A2 VIII Dose-Response Curves with Recombinant CYP3A4 and Human Liver Microsomes VividTM 3A4 Blue Oxprenolol Nitrofurantoin Diphenylhydantoin 0 -9 -8 -7 -6 -5 -4 [Testosterone] log {M} 1A 2 2A 6 2B 6 2C 8 2C 2C 9 18 2C 19 2D 6 2E 1 3A 4 3A 5 O 0 -9 -8 -7 -6 -5 -4 1 0 O -9 -8 -7 -6 -5 -4 RHI Red Green Blue Cyan Isoniazid Retinoic acid Phenylbutazone When the assay is performed in competitive TM mode Vivid 3A4 Green metabolism by CYP3A4 is inhibited by fewer compounds than any of the other probe substrates. This permits distinction of turnover-dependent inhibitors from competitive inhibitors by a simple change in the assay protocol. Drugs were either pre-incubated under conditions permitting turnover (turnover), or added to the enzyme at the same time as the fluorogenic substrates (competitive). Drugs whose CYP3A4 inhibitory activity has a substantial turnoverdependent component are highlighted in bold. Shown is % inhibition at 10µM [drug] relative to 10µM ketoconazole (competitive mode) or 10µM ellipticine (turnover mode). Red Green Blue Cyan Red Green Blue Cyan D 100 75 HLM [Troleandomycin] log {M} HLM Doxorubicin Ticlopidine IX 0 -9 -8 -7 -6 -5 Tamoxifen 100 102 101 96 84 100 98 97 94 94 93 90 89 86 86 83 78 56 45 44 36 35 35 34 21 19 12 10 8 Turnover-dependence 75 [Ketoconazole] log {M} 1A 2 2A 6 2B 6 2C 8 2C 2C 9 18 2C 19 2D 6 2E 1 3A 4 3A 5 -1 v {min } O RHI 25 RHI Poten cy Red 100 50 C ked b y Shown are the % inhibition values for >100 drugs in TM 3A4 assays at 10µM [drug] using Vivid recombinant human CYP3A4. Drugs were ranked by TM potency in the Vivid 3A4 Red assay and the 15 most potently inhibiting drugs are indicated in black. VividTM 3A4 Red detects inhibitors ( ) and activators TM (off scale, not shown) of CYP3A4, Vivid 3A4 Green implicates a lesser number of competitive inhibitors (fewer black dots above 50% inhibition potency), while TM Vivid 3A4 Blue and Cyan detect additional drugs that interact with CYP3A4 (blue & cyan dots inside yellow oval). B Red Green Blue Cyan -9 -8 -7 -6 -5 VividTM 3A4 Green O 20 50 0 O 15 HLM Red Green Blue Cyan 1 4 10 BR: Benzylresorufin; DBF: Dibenzylfluorescein; BCC: 7-Benzyloxy-3-cyanocoumarin BFC: 7-Benzyloxy-4-trifluoromethylcoumarin 2 N 5 [substrate] {µM} Assays were performed with 5nM human CYP3A4 (insect cell microsomes containing baculovirus-expressed 3A4 and NADPH-P450 reductase; Panvera, WI). 75 Green s ran VividTM 3A4 Substrates Detect Drug-Substrate Interactions Kinetic properties of VividTM substrates (solid lines) were compared sideby-side with the structurally related substrates (dashed) and with ( , ) or without ( , ) cytochrome b5. Line color indicates the color of the fluorescent metabolite generated. Red fluorogenic substrates display Michaelis-Menten type kinetics while all other substrates show sigmoidal velocity curves with Hill coefficients ~2. % Activity VividTM 3A4 Red Drug 10 0 RHI 0 >100 CYP3A4 Substrate Kinetics 100 Ellipticine Mifepristone Asarinin Papaverine Verapamil Dicyclomine Erythromycin Clemastine Amiodarone Thioridazine Vinblastine Nortryptyline Clomipramine Disulfiram Omeprazole 20 20 40 A Dihydroergotamine Blue [substrate] {µM} V II 20 40 -20 0 0 60 Cyan VividTM 3A4 Cyan BCC H O 4 -1 40 H O O 2 30 60 -1 HO 10 v {min } R1 v {min } HO 0 0 R2 Nicardipine Clotrimazole Bromoergocryptine 98 98 95 93 71 40 22 -7 48 36 30 30 32 1 6 13 -3 0 -11 4 8 3 -9 -9 -1 -13 1 -9 21 X CYP2E1 % Inhibition -1 BR R1 DBF 5 80 turnover -1 O ABSTRACT 2 10 competitive v {min } H 4 Ketoconazole % Activity Kcal/mol H 15 4 O Kcal/mol 79.5 MODE DRUG 100 id TM 3A H H VII IV VividTM 3A4 Green 20 v {min } -1 v {min } 53.9 Here we present two diverse sets of fluorogenic substrates for CYP3A4 and 2C9. Vivid™ substrates are highly sensitive probes for drug interactions with these isozymes and permit assay miniaturization to < 2µl volumes and enable automated high throughput screening. This panel of sensitive CYP450 probes also permits the distinction of different modes of drug-CYP450 interactions. Competitive, noncompetitive, and activator interactions are readily distinguished. The large amount of information generated in high throughput assays is likely to speed the drug-lead optimization process and to give guidance to chemists in the development of leads that are less likely to fail because of drug interactions. VividTM 3A4 Red 6 TM Inhibitory interactions of drugs with metabolizing enzymes are a major cause for adversial drug-drug interactions. Testing compounds early in drug discovery for these unwanted interactions will permit elimination of unsuitable compounds and chemical series from further drug development. With the everexpanding pool of candidate drugs and increasing number of hits emerging from primary screening this task is becoming increasingly more laborious. To complicate matters, CYP3A4 and CYP2C9 display complex kinetic behaviors and inhibitory profiles that are dependent on the probe substrate used. This is due to their large active sites that may bind two substrates simultaneously. To obtain meaningful SAR data, several kinetically differing substrates are needed that sense drug-interactions at any of the metabolism-affecting sites in these isozymes. B A Viv III Potential Sites of Oxidation % Inhibition I References 1. Ekins, S. et al. (1998). Int. J. Clin. Pharmacol. Ther., 36, 642 2. Harlow, G.R. & Halpert, J.R. (1998). PNAS U S A, 95, 6636 3. Houston, J.B. & Kenworthy, K.E. (2000). Drug Metab. Dispos., 28, 246 4. Koley, A.P. et al. (1997). J. Biol. Chem., 272, 3149 5. Korzekwa, K.R. et al. (1998). Biochemistry, 37, 4137 6. Shou, M. et al. (1999). Biochem. J., 340, 845 7. Thummel, K.E. & Wilkinson, G.R. (1998). Annu. Rev. Pharmacol. Toxicol., 38, 389 8. Ueng, Y.F. et al. (1997). Biochemistry, 36, 370 Summary & Discussion Aurora's VividTM substrates are benzyloxymethyl and alkyloxymethyl derivatives of highly fluorescent phenolic dyes (Panel I). Initially, the ether substrates show little or no fluorescence. Metabolism by CYP450 isozymes liberates TM fluorescent dyes with high extinction coefficients and high aqueous fluorescence quantum yields permitting sensitive detection of CYP450 isozyme activity. The excellent kinetic behavior of the Vivid 3A4 substrates (Panel III) is possibly due to the increased freedom of rotation and movement of the methylenes carrying the abstractable hydrogens relative to the more rigid benzyl ether substrates. This may permit better access of these hydrogens to the heme in the CYP450 active site. As depicted in Panel I, abstraction of any one of the four of the aliphatic hydrogens in the benzyloxymethyl moiety leads to a fluorescent product, possibly increasing the probability of metabolism by CYP3A4 isozyme, which is known to metabolize substrates at multiple sites. TM TM Aurora has developed a diverse set of fluorogenic substrates for CYP3A4 (Panel II & III). Vivid 3A4 substrates show excellent to good selectivity for CYP3A isozymes permitting their use in human liver microsomal TM preparation as well as in recombinant CYP3A4 preparations (Panels II, V & VI). Vivid 3A4 substrates are highly sensitive probes for drug interactions with this isozyme and permit assay miniaturization to < 2µl volumes (Panel VIII) and enable automated high throughput screening. A benefit of this set of substrates is that it permits both, high throughput screening of entire compound collections using well defined recombinant enzyme preparations as well as permitting the follow-up analysis in human liver microsomal preparations using the same probe substrates (Panels IV, V & VI). VividTM 3A4 Red interacts with CYP3A4 in a manner that reports drugs that can partially inhibit the enzyme or cause enzyme activation (Panels V B, C & D) in the recombinant CYP450 enzyme system. In a screen of >100 drugs (Panel IV), VividTM 3A4 Blue and Cyan pick up additional inhibitory CYP450-drug interactions, while VividTM 3A4 Green is comparatively less sensitive. This property of VividTM 3A4 Green is useful in determining whether a drug is a turnover dependent inhibitor of CYP3A4 (Panel VII). In human liver microsomes, the situation is similarly complex, in that inhibitors have differential effect on the metabolism of VividTM 3A4 probe substrates (Panels V & VI). It has been shown that inhibition of drug probe metabolism is less pronounced in human liver microsomal preparations than in recombinant CYP450 isozyme preparations (7). Similarly, metabolism of VividTM 3A4 substrates is generally less affected by test compounds in human liver microsomal preparations (Panels V & VI). The spreadsheet (above) lists the mean apparent Ki values of inhibition of CYP3A4 by a panel of drugs as assessed by their TM inhibition of Vivid 3A4 substrate metabolism. The source of CYP3A4 activity was either from recombinant human isozyme (RHI) preparations or pooled human liver microsomes (HLM). Shown for comparison are value ranges from the recent literature for inhibition of CYP3A4 drug substrate metabolism by human liver microsomes. AuroraTM has also developed a diverse set of fluorogenic substrates for 2C9 (kinetics: Panel IX). VividTM 2C9 Blue interacts with CYP2C9 in a manner that reports drugs that can partially inhibit the enzyme or cause enzyme activation (Panels X B & C) in recombinant isozyme preparations, while VividTM 2C9 Red and Green experience full inhibition, though at different drug dosages. Potent CYP2C9 inhibitors that were present in a set of > 100 drugs TM were found with all three Vivid 2C9 substrates (Panel XII). Lit. K: Thummel, K.E. & Wilkinson, G.R. (1998). Annu. Rev. Pharmacol. Toxicol., 38, 389. Poster presented at Gordon Research Conference on Drug Metabolism, Holderness School, Plymouth NH, July 9-14, 2000.