Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

Brandt, Richard B.; Laux, Jerome E.; Yates, S. W.

doi:10.1016/0885-4505(87)90046-6

Cited by 68 publications

(46 citation statements)

References 5 publications

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“…For a competitive inhibitor, the IC 50 is expected be the same as the K i when the substrate is << the K m . 21 The Ac-DEVD-CHO inhibitor titration indicated an IC 50 of 140 pM (Fig. 8A); this was very close to the published K i of 230 pM.…”

Section: Determination Of Ic 50 and Ec 50 Valuessupporting

confidence: 82%

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

et al. 2005

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Section: Determination Of Ic 50 and Ec 50 Valuessupporting

confidence: 82%

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

et al. 2005

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“…(1) and (2)) and general properties of MM approximation (Brandt et al, 1987;Yung-Chi and Prusoff, 1973) (Santiskulvong et al, 2011), is almost independent of cell lines sensitive to RAD001 and satisfactorily correlates with the dissociation constant and IC 50 values obtained in the modelling.…”

Section: Discussionsupporting

confidence: 73%

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

Goltsov

Tashkandi

Langdon

et al. 2017

European Journal of Pharmaceutical Sciences

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The phosphatidylinositide 3-kinases (PI3K) and mammalian target of rapamycin-1 (mTOR1) are two key targets for anti-cancer therapy. Predicting the response of the PI3K/AKT/mTOR1 signalling pathway to targeted therapy is made difficult because of network complexities. Systems biology models can help explore those complexities but the value of such models is dependent on accurate parameterisation. Motivated by a need to increase accuracy in kinetic parameter estimation, and therefore the predictive power of the model, we present a framework to integrate kinetic data from enzyme assays into a unified enzyme kinetic model. We present exemplar kinetic models of PI3K and mTOR1, calibrated on in vitro enzyme data and founded on Michaelis-Menten (MM) approximation. We describe the effects of an allosteric mTOR1 inhibitor (Rapamycin) and ATP-competitive inhibitors (BEZ2235 and LY294002) that show dual inhibition of mTOR1 and PI3K. We also model the kinetics of phosphatase and tensin homolog (PTEN), which modulates sensitivity of the PI3K/AKT/mTOR1 pathway to these drugs. Model validation with independent data sets allows investigation of enzyme function and drug dose dependencies in a wide range of experimental conditions. Modelling of the mTOR1 kinetics showed that Rapamycin has an IC 50 independent of ATP concentration and that it is a selective inhibitor of mTOR1 substrates S6K1 and 4EBP1: it retains 40% of mTOR1 activity relative to 4EBP1 phosphorylation and inhibits completely S6K1 activity. For the dual ATP-competitive inhibitors of mTOR1 and PI3K, LY294002 and BEZ235, we derived the dependence of the IC 50 on ATP concentration that allows prediction of the IC 50 at different ATP concentrations in enzyme and cellular assays. Comparison of the drug effectiveness in enzyme and cellular assays showed that some features of these drugs arise from signalling modulation beyond the on-target action and MM approximation and require a systems-level consideration of the whole PI3K/PTEN/AKT/mTOR1 network in order to understand mechanisms of drug sensitivity and resistance in different cancer cell lines. We suggest that using these models in systems biology investigation of the PI3K/AKT/mTOR1 signalling in cancer cells can bridge the gap between direct drug target action and the therapeutic response to these drugs and their combinations.

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“…The relationship between K i and values of IC 50 determined when [S] = K m depends on the mechanism of inhibition, as summarized in Table 1 (Cheng and Prusoff , 1973;Brandt et al, 1987;Cer et al, 2009 …”

Section: Dmd #66597mentioning

confidence: 99%

The Reliability of Estimating K_i Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC₅₀ Values: A Retrospective Analysis of 343 Experiments

et al. 2015

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In the present study, we conducted a retrospective analysis of 343 in vitro experiments to ascertain whether observed (experimentally determined) values of Ki for reversible cytochrome P450 (P450) inhibition could be reliably predicted by dividing the corresponding IC₅₀ values by two, based on the relationship (for competitive inhibition) in which Ki = IC₅₀/2 when [S] (substrate concentration) = Km (Michaelis-Menten constant). Values of Ki and IC₅₀ were determined under the following conditions: 1) the concentration of P450 marker substrate, [S], was equal to Km (for IC₅₀ determinations) and spanned Km (for Ki determinations); 2) the substrate incubation time was short (5 minutes) to minimize metabolism-dependent inhibition and inhibitor depletion; and 3) the concentration of human liver microsomes was low (0.1 mg/ml or less) to maximize the unbound fraction of inhibitor. Under these conditions, predicted Ki values, based on IC₅₀/2, correlated strongly with experimentally observed Ki determinations [r = 0.940; average fold error (AFE) = 1.10]. Of the 343 predicted Ki values, 316 (92%) were within a factor of 2 of the experimentally determined Ki values, and only one value fell outside a 3-fold range. In the case of noncompetitive inhibitors, Ki values predicted from IC₅₀/2 values were overestimated by a factor of nearly 2 (AFE = 1.85; n = 13), which is to be expected because, for noncompetitive inhibition, Ki = IC₅₀ (not IC₅₀/2). The results suggest that, under appropriate experimental conditions with the substrate concentration equal to Km, values of Ki for direct, reversible inhibition can be reliably estimated from values of IC₅₀/2.

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Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

Cited by 68 publications

References 5 publications

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The Reliability of Estimating K_i Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC₅₀ Values: A Retrospective Analysis of 343 Experiments

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Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

Cited by 68 publications

References 5 publications

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model

Kinetic modelling of in vitro data of PI3K, mTOR1, PTEN enzymes and on-target inhibitors Rapamycin, BEZ235, and LY294002

The Reliability of Estimating Ki Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC50 Values: A Retrospective Analysis of 343 Experiments

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The Reliability of Estimating K_i Values for Direct, Reversible Inhibition of Cytochrome P450 Enzymes from Corresponding IC₅₀ Values: A Retrospective Analysis of 343 Experiments