ACS Medicinal Chemistry Letters
Page 4 of 7
eral, in the absence of H2O2, inhibitors provoked inactiva-
1
tion up to 19%, whereas hydrogen peroxide alone mini-
mally affected the activity (table 1). In the presence of
hydrogen peroxide 13A-1C, and 2C-, 3C-based ligands the
activity decreased by 10-15%. By contrast, 1A-1C, 6A-1C
and 13B-1C were able to cause an almost complete inhibi-
tion of MPO in the presence of hydrogen peroxide (resid-
ual activity in the range 18-25%, table 1). Interestingly,
these levels of inhibition correlated well with the predict-
ed models. Indeed, the docking poses of hydralazine- and
isoniazid-based hydrazones underline the important role
played by the nitrogen heterocycles of 1C and 3C, with
hydralazine being a better inhibitor than isoniazid.
Hence, in most of the molecules, the aromatic groups
governed interactions with the heme but additional con-
tacts due to aldehyde moieties seemed to lock the system.
This aspect is probably responsible for the higher affinity
and inhibition rate induced by ligands derived from hy-
dralazine. Moreover, the variation of inhibitory effect
between 1A-1C, 6A-1C, 13A-1C and 13B-1C might be re-
flected by the distance between hydralazine and heme
groups (Figure 3). Hydrazone 13A-1C acted as a reversible
inhibitor while 1A-1C, 6A-1C and 13B-1C were irreversible.
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C
The mechanism of MPO inhibition was subsequently
investigated by the multi-mixing stopped-flow technique.
Native ferric MPO [Fe(III)···Por] is oxidized (k1) by H2O2,
producing water and Compound I {oxoiron(IV) com-
bined with a porphyrin cation radical: [+•PorFe(IV)=O]}
(Figure 4). In the halogenation cycle, Compound I is
directly reduced back to the resting state (k2) by chloride
[or other (pseudo)halides], thereby releasing hypo-
chlorous acid. Alternatively, in the presence of one-
electron donors, the peroxidase pathway is followed, in-
Figure 4. Reactions catalyzed by human myeloperoxidase. In
the halogenation cycle Compound I is reduced by halides
(X-) directly to the ferric state [thereby releasing hypohalous
acids (HOX)], whereas in the peroxidase cycle Compound I is
reduced in two one-electron steps via Compound II to the
resting state (A). Reaction of MPO Compound I with 20 µM
13B-1C (B). Reaction of MPO Compound I with 500 µM 13B-
1C (C). Red spectra correspond to Compound II formation;
light blue spectra correspond to Compound III formation
and light green spectra show the decay to incomplete ferric
MPO. Insets show the time traces at 430 and 456 nm.
cluding Compound
I reduction to Compound II
[PorFe(IV)–OH] (k3), and Compound II reduction to the
ferric state (k4) (Figure 4).14 Here, Compound I reduction
was evaluated with the following ligands: 1A-1C, 13A-1C
and 13B-1C. In all cases, a direct and fast transition of
Compound I to Compound II (Soret maximum at 456
nm) was observed with clear isosbestic points (see Figure
4 middle ESI). The determined values of k3 were 2.8 × 105,
7.1 × 105, and 3.3 × 105 M–1 s–1 for 1A-1C, 13A-1C and 13B-1C,
respectively. The results clearly indicated that all selected
molecules behaved as good one-electron donors of Com-
pound I reacting similar to hydralazine alone (k3= 7.1 ×
105 M–1 s–1). In contrast, the reaction of the tested mole-
cules with Compound II gave variable results. Hydra-
zone 13A-1C showed a direct transition of Compound II
to ferric MPO (Soret maximum at 429 nm) with clear
isosbestic points, but this reaction was slow. The apparent
bimolecular rate constant (k4) was found to be 89.3 M-1 s-1,
indicating a high k3/k4 index of 7951. This high index sug-
gested that inhibitor 13A-1C induced the (reversible) ac-
cumulation of Compound II, which is outside the halo-
genation cycle (Figure 4). By contrast, during the reac-
tions of hydralazine and substrates 1A-1C and 13B-1C with
Compound II, a steady-state shift to Compound III was
observed (Figure 4 bottom). Compound III can only be
formed from ferric or ferrous MPO with activated oxygen
Compounds 1A-1C, 6A-1C and 13B-1C were predicted to
stack on the active site of MPO through the aromatic ring
of hydralazine, as seen on figure 3. In contrast, the dock-
ing pose with ligand 13A-1C emphasized an interaction
involving the aromatic group of 13A (Figure 3A).
For the sake of comparison, additional docking experi-
ments were carried out with ligands based on pharmaco-
phores 2C and 3C. Interestingly, all ligands derived from
isoniazid 3C make a π-π stacking with the heme of the
enzyme through the pyridyl group of 3C. In opposite,
hydrazones formed from 2C showed stacking poses with
the π-system of the aldehydes, except for 13A-2C since no
interaction was observed (see ESI). According to the pre-
dicted models, molecules bearing both aromatic and
polar functionalities have a greater binding affinity to-
ward MPO and therefore a higher inhibition ability.
Finally, we elucidated the mechanism of action of these
hydrazones and their capacity to act as irreversible inhibi-
tors. These experiments were performed in the presence
and absence of hydrogen peroxide, which is necessary to
initiate the catalytic cycle of MPO. In practice, mixtures
of ligands and enzymes were diluted 100-fold, followed by
measurement of the residual enzymatic activity. In gen-
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