Time-dependence of inhibition

Schwab BW, Richardson RJ. 1986. Lymphocyte and brain neurotoxic esterase Dose and time dependence of inhibition in the hen examined with three organophosphorus esters. Toxicol Appl Pharmacol 83 1-9. 

Failure to properly account for the time dependence of inhibition can result in grossly misleading SAR and potentially cause the researcher to overlook promising inhibitor molecules. 

Fig. 7.3. Time dependence of inhibition calculated from Escherichia coli biosensor exposure...

We have already used the interactions of methotrexate with dihydrofolate reductase (DHFR) several times within this text to illustrate some key aspects of enzyme inhibition. The reader will recall that methotrexate binds to both the free enzyme and the enzyme-NADPH binary complex but displays much greater affinity for the latter species. The time dependence of methotrexate binding to bacterial DHFR was studied by Williams et al. (1979) under conditions of saturating [NADPH], In the presence of varying concentrations of methotrexate, the progress curves for DHFR activity became progressively more nonlinear (Figure 6.14). The value of kobs from... 

Adsorption versus Polymerization. It is instructive to examine further the time dependence of the corrosion inhibition. In acid corrosion inhibition tests, steady state is customarily assumed to be reached within 10 to 20 min after initial exposure of the metal specimen. Since the inhibitors function by reducing the available active surface area, we expect an increase in and a corresponding decrease in P. The degree of corrosion protection the inhibitor provides is given by... 

An adjacent tnfluoromethyl group sharply increases the electrophilic character of the carbonyl carbon Compounds that readily form hydrates and hemiacetals show a time-dependent reversible inhibition of the enzyme acetylcholinesterase (equation 2), in which the tight complex makes inhibition only partially reversible [75] In comparison with a nonfluonnated analogue, several aliphatic ketones flanked by CF3 and CF2 groups, are exceptionally potent reversible inhibitors of acetylcholinesterase, as documented by comparison of inhibition constants Kv shown in equation 3 [76]... 

A was supported by a 3D X-ray structure of the inhibitor at the closely related porcine elastase (Navia, 1987) (Figure 13.12). After initially reversible inhibition (Figure 13.12, top two rows), time-dependent irreversible inhibition through covalent bonding of the dihydrothiazine ring of the inhibitor to the His-57 residue of the active site of the enzyme increasingly takes over (Figure 13.12, bottom row). Peptidyl chloromethyl ketones, known unspecific inhibitors, act in similar fashion. 

An important characteristic of inhibition of drug metabolism by an inactivator is the time dependence of both the onset and offset of the effect. The time course of the change in enzyme concentration from the baseline, Ess, to that in the presence of inactivator, Erss, is given by... 

FIGURE 31.6 PET scans showing dose dependency and time dependency of lazabemide inhibition of monoamine oxidase, type B in human brain. (Reproduced with permission from Fowler JS, Volkow ND, Wang G-J, Dewey SL. J Nucl Med 1999 40 1154-63.)... 

Enzyme inhibition data are often presented as IC50, the concentration of the inhibitor to cause 50 percent inhibition at one chosen substrate concentration Kt, the inhibition constant (dissociation constant from the inhibitor-enzyme complex) determined by enzyme kinetic analysis (e.g., Dixon plot) and /Cin lcl, the time-dependent inhibition constant for mechanism-based inhibitors. IC50 values can be estimated from the study described earlier. A positive inhibition, defined as dose-dependent inhibition, with the inhibited activity lower than 50 percent of that of the negative control, will require further experimentation to define Ki for a better evaluation of in vivo inhibitory potential. Further, a study to determine Klwul may be performed to evaluate if the inhibitor acts via covalent binding to the active site of the enzyme, leading to time-dependent irreversible inhibition. 

Zhang, F., Zu, S., and Iadecola, C., Time dependence of nitric oxide synthase inhibition on cerebral ischaemic damage, J. Cereb. Blood Flow Metab., 15, 595, 1995. 

To evaluate further the dose and time dependence of the blood NTE assay, we studied NTE inhibition in brain and blood short time (4 hr) after dosing hens with PrDChVP (0.32 - 1.0 mg/kg, im), as well as 24, 48, 72 and 96 hr after the maximal injected dose, 1.0 mg/kg [98], Brain and blood NTE were inhibited in a dose-responsive manner 4 hr after dosing (linear trend, P < 0.0001), and NTE inhibition was highly correlated between brain and blood (r = 0.997). 

In the reverse reaction, driving the synthesis of S3P and PEP from EPSP and phosphate, the amplitude of the product formation was altered because of the formation of a dead-end complex with S3P and phosphate bound to the active site. The time dependence of this inhibition during the single turnover to form E-S3P would lead to a model too complex to solve explicitly. The reaction time... 

It was noted that the 19-hydroxy- (33) and 19-oxo-3-methylene (34) analogues exhibited time-dependent irreversible inhibition of AR while the corresponding 10-methyl did not, suggesting that functionalization of the 10-methyl was necessary for the enzymatic activated inhibition. 

Because of the experimental manifestation of time-dependency and requirement for CYP catalysis, mechanism-based CYP inactivation is often referred to as time-dependent, metabolism-dependent, or preincubation-dependent inhibition. A detailed description of the kinetic characteristics of this type of inhibition has been published (Silverman, 1988) and a simplified kinetic equation is presented in Table 5.1. In cases where CYP activity can be recovered by dialysis, the term quasi-irreversible inhibition has been proposed (Ma et al., 2000). In addition, a time-dependency of CYP inhibition can result from the formation of potent yet reversible metabolites (Ma et al., 2000 Zhao et al., 2002 Zhout et al., 2005). Formation of a metabolite-intermediate (MI) complex has been described as another cause for time-dependent CYP inhibition by many quasi-irreversible inhibitors in this situation the metabolite or intermediate coordinates with the heme-ion thus decreasing the rate of catalysis. 

Parallel pathways of drug elimination Time-dependent CYP inhibition... 

Light emitted by chemiluminescent substrate was expressed in relative light units (RLU). The time dependence of light intensity was measured and the peak intensity was converted into percent of inhibition relative to control. We expressed the results as total antioxidant potential, expressed as percent inhibition TAPcl = ((max RLUconiroi) - (max RLUsampie)) 100 / (max RLUcontroi). Figure 1 shows the correlation between sum of phenolic compounds... 

Although one of the more complex electrochemical techniques [1], cyclic voltammetry is very frequently used because it offers a wealth of experimental information and insights into both the kinetic and thermodynamic details of many chemical systems [2], Excellent review articles [3] and textbooks partially [4] or entirely [2, 5] dedicated to the fundamental aspects and apphcations of cyclic voltammetry have appeared. Because of significant advances in the theoretical understanding of the technique today, even complex chemical systems such as electrodes modified with film or particulate deposits may be studied quantitatively by cyclic voltammetry. In early electrochemical work, measurements were usually undertaken under equilibrium conditions (potentiometry) [6] where extremely accurate measurements of thermodynamic properties are possible. However, it was soon realised that the time dependence of signals can provide useful kinetic data [7]. Many early voltammet-ric studies were conducted on solid electrodes made from metals such as gold or platinum. However, the complexity of the chemical processes at the interface between solid metals and aqueous electrolytes inhibited the rapid development of novel transient methods. 

Fig. 4. The time dependence of cell-free enzyme synthesis uninhibited and inhibited at varying times by actinomycin or rifampicin. Synthesis was carried out as described in Table 1 except for the following modifications. In the control experiment, samples were removed from the synthesis system and assayed at the times indicated on the abscissa. In the actinomycin and rifampicin experiments, the inhibitor (2 jitg of actinomycin or 1 ju,g of rifampicin per milliliter) was added at the time indicated on the abscissa, and the incubation was continued for another 60 minutes before assay. All values have been normalized to an assay time of 200 minutes. The S-30 used was prepared from strain 514 which contains a deletion of the entire lac region including the repressor gene.

The COX-2-selective diarylheterocycHc inhibitors have been reported to be a reversible competitive inhibitor of COX-1 while demonstrating time-dependent irreversible inhibition of COX-2 which accounts for the potency and selectivity demonstrated by members of this structural class [229]. The phenylsulphonamide moiety of the diarylheterocycles associate within a side pocket present in the active site of COX-2 and this pocket is more accessible in COX-2 than in COX-1, which is the result of the substitution of a vahne for an isoleucine foimd at position 523 in COX-1 [235]. 

The time dependence of the inhibition usually follows a first-order rate law. Epoxide concentrations necessary to achieve more than 98% inhibition in about 8 hr are in the range of 0.2 to 2 mAf (40 mM with sucrase-isomaltase ). Deviations may occur that are due to an enzyme-catalyzed reaction of the epoxide with halide ions in the medium high concentrations of these ions should be avoided. If salt is necessary to keep the enzyme in solution, sodium perchlorate or another salt with a nonnucleophilic anion should be used. 

Inhibition of Glucosidases with Bromoconduritol. a-Glucosidase from yeasf and /8-glucosidases from Cicer arietum and Alocasia macrorrhiza show a time-dependent, irreversible inhibition with very low concentrations of bromoconduritol. Prom experiments with preparations partially enriched with respect to A or B, it was found that isomer A reacts with a-glucosidase whereas the above 8-glucosidases react preferentially with B. 

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