Substrate probes

DNA microarrays, or DNA chips consist of thousands of individual DNA sequences arrayed at a high density on a single matrix, usually glass slides or quartz wafers, but sometimes on nylon substrates. Probes with known identity are used to determine complementary binding, thus allowing the analysis of gene expression, DNA sequence variation or protein levels in a highly parallel format. 

By using an identical radical clock substrate probe which did not rearrange upon hydroxylation with M. capsulatus (Bath), rearranged product was detected with MMO from AT. trichosporium 0B3b (59). From the ratio of unrearranged to rearranged products, a rebound rate constant was calculated to be 6 x 1012 s-1 at 30°C for this system. A separate study with another radical clock substrate probe with MMO from M. trichosporium 0B3b reported products consistent with both radical and cationic substrate intermediates (88). 

Since work with the radical clock substrate probes indicated important differences in the hydroxylation mechanisms for M. capsulatus (Bath) and M. trickosporium OB3b, work with (R) and (S)-[1-2H,1-3H]ethane with both enzymes was carried out (93, 94). With M. tri-chosporium OB3b, approximately 65% of the product displays retention of stereochemistry (93). A rebound rate constant of 2 - 6 x 1012 s-1 was calculated, assuming a free energy change of 0.5 kcal mole-1 for rotation about the C-C bond (94). This estimate approaches the value obtained from the radical clock substrate probe analysis (59). 

In summary, mechanistic studies have revealed intriguing differences between MMO from M. capsulatus (Bath) and MMO from M. triehosporium OB3b. With M. capsulatus (Bath), radical clock substrate probes indicated either that a substrate radical is not produced or that it reacts with a rate constant > 1013 s-1. With MMO from M. triehosporium OB3b, radical involvement was suggested from several experiments, and a rebound rate constant of 6 x 1012 s"1 was calculated for this system. 

Imaging can also be useful for multiprobe detection, for example using fluorescent probes together with CL reactions. Potentially it is possible to detect first a fluorescent probe, and second detect the CL probe by adding the CL substrate. Probes marked with different CL labels, usually enzymes requiring different substrates, can also be used at the same time, provided the first CL substrate is removed before a second CL analysis is performed by adding another substrate. 

Bush ED, Trager WF. Substrate probes for the mechanism of aromatic hydroxylation catalyzed by cytochrome P-450 selectively deuterated analogues of warfarin. J Med Chem 1985 28(8) 992-996. 

Darbyshire JF, Iyer KR, Grogan J, et al. Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450. Drug Metab Dispos 1996 24(9) 1038-1045. 

Gentest (now BD Biosciences) was the first to develop spectrophotometric assays to study CYP inhibition [98]. These assays are based on the turnover of mildly fluorescent substrate probes to moderately fluorescent metabolites, where metabolite formation is monitored by an increase in fluorescence using a plate reader [99,100]. Problems with these methods include background interference due to low signal-to-noise ratio, chemotype-specific interference and fluorescence quenching. Aurora Biosciences (now Vertex) has designed probes that exhibit larger fluorescence... 

Figure 9.3 The effects of varying levels of accessory proteins on CYP2C9 kinetics using diclofenac (a) or (S)-warfarin (b) as substrate probe [219].

Partial inhibition occurs when an inhibitor partially inhibits the turnover of a substrate, but is not able to fully inhibit substrate turnover even at saturating conditions [143], The most common explanation for partial inhibition is simultaneous occupancy of the CYP active site by both the effector and substrate probe in... 

Most likely, both observations are valid, depending upon substrate, probe, and printing conditions. 

Characterization of CYP2B6 protein levels in human liver has been somewhat problematic. Many antibody preparations prepared against rodent CYP2B forms appear to cross-react poorly with the human form. In addition, a CYP2B6-specific or CYP2B6-selective substrate probe has not yet been identified. Therefore, the tools for analysis of human CYP2B6 expression are not fully available. 

Several studies have been carried out using different substrate probes to determine the inhibitory potency of various members of this class against CYP2D6 (102-105). The potential implications of CYP2D6 (and other P450 enzymes) inhibition by this class of dmgs has been exhaustively reviewed (106-116) and is not considered further here. 

Krishnaswamy S, Duan SX, von Moltke LL, et al. Validation of serotonin (5-hydroxtryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6. Drug Metab Dispos 2003 31(1) 133 139. 

Lucas D, Menez JF, Berthou F. Chlorzoxazone an in vitro and in vivo substrate probe for liver CYP2E1. In Johnson EF, Waterman MR, eds. Cytochrome P450, Part B Methods in Enzymology. San Diego, CA Academic Press, 1996 272 115-123. 

Liu, K. E., Johnson, C. C., Newcomb, M., and Lippard, S. J., 1993, Radical clock substrate probes and kinetic isotope effect studies of the hydroxylation of hydrocarbons by methane monooxygenase, J. Am. Chem. Soc. 115 939n947. 

Two types of substrate probe, cis- and trans-l,3-dimethylcyclopentane and ethyl- and 2-propylcyclopropane, were used to shed light on mechanishc details of the hydroxylahon step [30]. In the use of the first two probes, the participation of Ti—OOH species in a concerted mechanism would predict either the retention or the inversion of configurahon at the chiral center, while the stereochemistry of a homolytic mechanism would be determined by the compehhon between the epimerization of the transient terhary carbon radical and C—O formation (Scheme... 

Decay of substrate, probing compound, or oxygen consumption... 

Substrate Probe n-Octane Butanol Butyl amine Ethylene Diamine... 

Figure 16a and b shows the effect of L on the radial dependence of the steady-state concentration and flux at the substrate/solution interface for a first-order dissolution process characterized by Ki = 10 and L = 0.1, 0.32, and 1.0. As the tip-substrate separation decreases, the effective rate of diffusion between the probe and the surface increases, forcing the crystal/so-lution interface to become more undersaturated. Conversely, as the UME is retracted from the substrate, the interfacial undersaturation approaches the saturated value, since the solution mass transfer coefficient decreases compared to the first-order dissolution rate constant. Movement of the tip electrode away from the substrate also has the effect of promoting radial diffusion, and consequently the area of the substrate probed by the UME increases. 

Some commercially available phenotyping kits use an array of coumarin analogs designed to be relatively isozyme-specific substrates (probes) that are metabolized to products with easily measurable spectral characteristics. Other commercially available kits use microsomes from baculovirus-infected cells that overexpress individual human GYP isoforms and fluorescent substrates (Vivid substrates) that can be incorporated into 1536 well formats. These simple systems do not readily lend themselves to the in vitro study of enzyme induction, however. The prediction of xenobiotic alteration of the expression of GYP activity in vivo from in vitro experiments will be discussed more completely in the chapter on drug—drug interactions. 

Examples of Substrates (Probes) Used for In Vitro Phenotyping of GYP Activity... 

Brazeau, B.J., R.N. Austin, C. Tarr, J.T. Groves, and J.D. Lipscomb (2001). Intermediate Q from soluble methane monooxygenase hydroxylates the mechanistic substrate probe norcarane Evidence for a stepwise reaction. J Am. Chem. Soc. 123, 11831-11837. 

Figure 2.15. (a) The rebound mechanism, (b) Derivation of the apparent lifetime (r pP of a putative radical intermediate from the ratio of rearranged (R) to unrearranged (U) alcohol products produced from P450 hydroxylation of a substrate probe. 

F.J. Gonzalez, and J.O. Miners (1993). Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques. Biochem. Pharmacol. 46, 1975-1981. 

Miners, J.O. and D.J. Birkett (1996). Use of tolbutamide as a substrate probe for human hepatic cytochrome P450 2C9. Meth. Enzymol. 272, 139-145. 

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