An algebraic model for the time-dependence of IC50 in covalent enzyme inhibition assays

Petr Kuzmič
BioKin Ltd., Watertown, Massachusetts, USA
http:// www.biokin.com

Abstract
[...]
Key words: [...]

1. Introduction where the enzyme and inhibitor were pre-incubated for various
lengths of time in the absence of substrate. At the end of the
[...] pre-incubation period, the substrate is added and initial reaction
velocity is determined as a measure of the residual enzymatic
2. Methods activity. This is the type of experiment conducted by Kitz &
Wilson in their seminal paper on covalent inhibition [4]. For
This section describes the theoretical and mathematical meth- a more recent example see ref. [5]. The details of algebraic
ods that were used in heuristic simulations described in this re- derivations are shown in the Supporting Information.
port. All computations were performed by using the software
package DynaFit [1, 2]. 2.2.1. Two-step kinetic mechanism “B”
Under the assumption that a given inhibitor follows the two-
2.1. Kinetic mechanisms of irreversible inhibition step kinetic mechanism “B”, the relationship between the ob-
In this report we will consider in various contexts the kinet- served I50 and the underlying kinetic constants is expressed by
ics mechanisms of substrate catalysis and irreversible inhibition the implicit algebraic Eqn (1). Note that the numerical (itera-
depicted in Figure 1. For further details see ref. [3]. tive) solution can only be obtained by the bisection method [6,
p. 350]; attempts to solve for I50 in terms of kinact , Ki∗ , and t50
k1s k2s by using either the Newton-Raphson method [6, p. 362] or the
E+S E•S E+P fixed-point iteration method [7] failed. The “true” value of the
k-1s inhibition constant Ki can be computed from the “apparent” Ki∗
by using Eqn (2).
k1 k2 ( )
"A": E+I E•I EI I50 I50
k-1 0 = 1+ − 2 exp − k t (1)
Ki∗ I50 + Ki∗
inact 50

( )−1
Ki k2 [S]0
Ki = Ki∗ 1+ (2)
"B": E+I E•I EI KM
Ki = k-1 / k1 It is interesting to compare Eqn (1) to a similar implicit
equation Eqn (3) previously derived by Krippendorff et al. [8].
k1 Note that Krippendorff’s Eqn (3) applies to an experiment where
"C": E+I EI the enzyme, the substrate, and the covalent inhibitor are brought
into contact immediately form the start of the assay and the I50
Figure 1: Kinetic mechanisms of substrate catalysis (top) and
is determined with respect to the concentration of product P
covalent inhibition (mechanisms A – C).
formed at the stopping time t50 , as opposed to with respect to
the residual enzyme activity.

2.2. Mathematical models ( )

I50 I50
The following mathematical models apply for the relation- 0 = 1− kinact t50 − exp − kinact t50 (3)
ship between the I50 and kinetic constants in an experiment 2 Ki∗ I50 + Ki∗

BioKin Technical Note TN-2020-04 :: D RAFT Revision 1.03 :: 18 July 2020

2.2.2. One-step kinetic mechanism “C”
Under the assumption that a given inhibitor follows the one-
step kinetic mechanism “C”, the relationship between the ob-
served I50 and the underlying value of 1˛∗ is expressed by Eqn
(4), where ln(2) = 0.693. Note that an essential equation was
initially derived, in different context, by Maurer et al. [9]. The
“true” value of the bimolecular association rate constant k1 can
be computed from the “apparent” value k1∗ by using Eqn (5).
ln(2)
k1∗ = (4)
I50 t50
( )
[S]0
k1 = k1∗ 1 + (5)
KM
It is interesting to compare Eqn (4) to a similar equation
Eqn (6), which applies to an experiment where the enzyme, the
substrate, and the covalent inhibitor are brought into contact
immediately form the start of the assay. See ref. [10] for details.
[4] R. Kitz, I. B. Wilson, Esters of methanesulfonic acid
as irreversible inhibitors of acetylcholinesterase, J. Biol.
Chem. 237 (1962) 3245–3249.
URL https://www.jbc.org/content/237/10/3245.long
[5] F. Huang, H. Hu, K. Wang, C. Peng, W. Xu, Y. Zhang,
J. Gao, Y. Liu, H. Zhou, R. Huang, M. Li, J. Shen, Y. Xu,
Identification of highly selective lipoprotein-associated
phospholipase A2 (Lp-PLA2) inhibitors by a covalent
fragment-based approach, J. Med. Chem. 63 (13) (2020)
7052–7065.
URL https://doi.org/10.1021/acs.jmedchem.0c00372
[6] W. H. Press, S. A. Teukolsky, W. T. Vetterling, B. P.
Flannery, Numerical Recipes in C, Cambridge University
Press, Cambridge, 1992.
[7] P. Kuzmič, Fixed-point methods for computing the equi-
librium composition of complex biochemical mixtures,
Biochemical J. 331 (1998) 571–575.
URL https://doi.org/10.1042/bj3310571

1.5936 [8] B.-F. Krippendorff, R. Neuhaus, P. Lienau, A. Reichel,

k1∗ = (6) W. Huisinga, Mechanism-based inhibition: Deriving KI
I50 t50
and kinact directly from time-dependent IC50 values, J.
Biomol. Screen. 14 (2009) 913–923.
3. Results
URL https://doi.org/10.1177/1087057109336751
[...]
[9] T. Maurer, M. Tabrizi-Fard, H. Fung, Impact of
mechanism-based enzyme inactivation on inhibitor po-
4. Discussion tency: implications for rational drug discovery, J. Pharm.
Sci. 89 (2000) 1404–1414.
[...] URL https://bit.ly/39adW3D

[10] P. Kuzmič, A two-point IC50 method for evaluating the

Acknowledgements biochemical potency of irreversible enzyme inhibitors,
[...] BioRxiv (2020) doi:10.1101/2020.06.25.171207.
URL https://doi.org/10.1101/2020.06.25.171207

Supporting information

[...]

References

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URL http://doi.org/10.1006/abio.1996.0238
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URL http://doi.org/10.1016/S0076-6879(09)67010-5

[3] P. Kuzmič, Deciding between one-step and two-step

irreversible inhibition mechanisms on the basis of
“kobs ” data: A statistical approach, BioRxiv (2020)
doi:10.1101/2020.06.08.140160.
URL https://doi.org/10.1101/2020.06.08.140160