Kinetics time-dependent inhibition

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. 

Fig. 4 Mechanism of action (MOA) and inhibition studies of ML119 (compound 1) with HePTP and HePTP mutants, (a) Progress curves of HePTP (6.25 nM) activity in the presence of different doses of compound 1 (0, 0.078,0.156,0.313,0.625,1.25 /jM) and 0.3 mM OMFP in 20 mM Bis-Tris, pH 6.0,150 mM NaCI, 1 mM DH, and 0.005 % Tween-20 in 20 /jL totai assay voiume in biack 384-weii microtiter plates. No time-dependent inhibition was observed as demonstrated by the linear progress curves of the HePTP phosphatase reaction, (b) Eadie-Hofstee plot of the Michaelis-Menten kinetic study with compound I.The HePTP-catalyzed hydrolysis of OMFP was assayed at room temperature in a 60 /jL 96-well format reaction system in 50 mM Bis-Tris, pH 6.0 assay buffer containing 1.7 mM DTT, 0.005 % Tween-20, and 5 % DMSO. Recombinant HePTP (5 nM) was preincubated with various fixed concentrations of inhibitor (0,0.1,0.2,0.4,0.8,1.6 /jM) for 10 min. The reaction was initiated by addition of various concentrations of substrate (0,12.5,25,50,100,200,400 pM) to the...

Little precise mechanistic studies have been undertaken with these inhibitors with the exception of the time-dependent inhibition of SAH hydrolase by 176. Stoichiometric loss of fluoride was observed by 19F-NMR during the inactivation process. However, there is only circumstantial evidence to support the addition-elimination mechanism proposed in Figure 1 all attempts to isolate an enzyme fragment covalently bound to an inhibitor have so far been unsuccessful. If the rate-determining step in the enzyme inhibition process is an attack of an enzyme nucleophilic residue on a 0-fluoro-a, 0-unsaturated imine or ketone, kinetic analysis of addition-elimination reactions to similar systems indicate that the ( )-isomer is the more active isomer (73) this could explain in part the isomeric preference seen with MAO and SAH hydrolase inhibitors. 

If the inhibitor combines irreversibly with the enzyme—for example, by covalent attachment—the kinetic pattern seen is like that of noncompetitive inhibition, because the net effect is a loss of active enzyme. Usually, this type of inhibition can be distinguished from the noncompetitive, reversible inhibition case since the reaction of I with E (and/or ES) is not instantaneous. Instead, there is a time-dependent decrease in enzymatic activity as E + I El proceeds, and the rate of this inactivation can be followed. Also, unlike reversible inhibitions, dilution or dialysis of the enzyme inhibitor solution does not dissociate the El complex and restore enzyme activity. 

More recent detailed kinetic investigations have revealed that actinonin is a time-dependent, essentially irreversible, inhibitor of PDF enzymatic activity [72]. This study demonstrated that the kinetics of inhibition of... 

The mechanisms by which an inhibitor adds to an oxidized hydrocarbon exerts its influence may differ depending on the reaction conditions. If the rate constants of the elementary reactions of RH, InH, R02 , In, ROOH, and 02 are known, the kinetics of the inhibited oxidation of RH can mathematically be described for any conditions. However, such an approach fails to answer questions how the mechanism of inhibited oxidation is related to the structure and reactivity of InH, RH, and R02 or what inhibitor appears the most efficient under the given conditions, and so on. At the same time, these questions can easily be clarified in terms of a topological approach whose basic ideas are the following [43-45,70-72] ... 

Interestingly, although many transition state analogs bind noncovalently to the target enzyme s active site via a one-step kinetic mechanism (Scheme la) and would therefore be expected to exhibit no time-dependent properties of inhibition, inhibitors with Kj values of < 10 10 M (like coformy-cin) usually have a slow onset of inhibition kobserved < 10 2 s 1 (i.e., an approach to equilibrium inhibition of > 1 min).161 This is merely an assay artifact due to... 

When data are available to enable comparison of the plasma concentration time profile after single administration with that after repeated administration, this would enable determination of whether the substance has time dependent kinetics (due to induction of metabolism, inhibition of metabolism, and/or accumulation and saturation of processes involved in distribution, metabohsm, and excretion). 

The industrial flow assurance paradigm is shifting from avoidance, enabled by thermodynamic inhibition, to risk management, enabled by application of kinetics. Examples of time-dependent flow assurance phenomena are kinetic inhibitors, AAs, plug dissociation, and electrical heating of pipelines for plug dissociation. Research support will move from thermodynamics, which is currently acceptably accurate for engineering applications, to time-dependent kinetics. 

All of the azides investigated were time-dependent inhibitors at millimolar concentrations and the inhibition was reversible in each case, with hepatic glutathione 5-transferase proving the most sensitive enzyme. Inhibitor potency appears to depend upon the substrate employed, -heptyl and allyl azides (60) and (62) being the most potent with NBC, and -butyl and -hexyl azide (57) and (59) when DNCB was included in the assay. Kinetic studies, where the GSH and DNCB concentrations were independently varied, indicated that compounds (61),(63) and (64) were noncompetitive inhibitors, while allyl azide (62) and the n-alkyl azides (56)-(60) inhibited the enzyme in a competitive manner. From these observations, the authors speculate that, in a process reminiscent of that known to occur with alkyl and aryl halides, glutathione 5-transferase may catalyse the conjugation of azides with GSH in vivo. 

Kinetics of inhibition by NSAIDs Immediate, competitive inhibition (strong for COX-1 inhibitors, weak for COX-2 inhibitors) COX-2 inhibitors noncompetitive (irreversible ), time-dependent COX-1 inhibitors competitive, immediate... 

The inhibition of pyruvate formate-lyase by hypophosphite was first observed by Novelli in work on the CoA-independent carboxylate exchange reaction between pyruvate and formate (186). In a more detailed study by Knappe et al., time-dependent inactivation is observed to occur with concomitant loss of the enzyme free radical EPR signal (180). The inactivation kinetics are first order and the rate of inactivation is accelerated when the enzyme is in the acetylated form. Furthermore, inactivation by [ HJhypophosphite leads to the stoichiometric release of tritium to H2O, and treatment of PFL with [ P]hypophosphite produces an alkali-labile radiolabel that is covalently bound to the inactive enzyme (180). 

In vitro studies with human term placental microsomes showed that PED displayed the typical properties of a mechanism-based inhibitor of cytochrome P450, namely (a) NADPH-dependent inhibition (b) time-dependent loss of enzymatic activity, followed by pseudo-first-order kinetics and (c) natural substrates delayed the irreversible inhibition. The irreversibility of inhibition was further substantiated by dialysis experiments ... 

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