Journal of Medicinal Chemistry
Article
IC50(cell): 0.74 μM]. The same gain in activity occurred for 8a
in cells [IC50(enz): 1.1 μM, IC50(cell): 0.02 μM] compared to 8.
STD-NMR Study of the Interaction between the Active
Site and the Inhibitor. To better understand the observed
SARs and notably how the nature and positioning of the lateral
N-sidechain influenced the binding of the inhibitor inside the
active pocket, we performed epitope mappings on several
couples of regioisomers using saturation transfer difference
NMR (STD-NMR). In STD-NMR, selective saturation of
hTDO2 in solution is transferred to the bound ligand through
intermolecular nuclear Overhauser effects (NOEs) between
protons of the inhibitor and the active site’s residues.
Consequently, the ligand’s protons interacting more closely
display intense STD signals, while a decrease in STD intensity
correlates with lower interaction.41 Due to solubility issues, we
focused our investigation on compounds bearing solubilizing
moieties to attain a high compound concentration (up to 200
μM) in aqueous buffer, suitable for the present evaluation. We
mapped the epitopes of regioisomers 25−26, 35−36, and 31−
32 (Figure 3). Epitope mapping of all six compounds suggested
that N3-substitutions led to an improper position of the indole
ring compared to N1-substitutions (data regarding compounds
Figure S2). Indeed, as pictured in Figure 3, epitope mapping on
the different compounds showed that the indole ring displays
the highest saturation transfer for protons H5, H6, and H7
(around 100%). Concurrently, cocrystallographic structures of
hTDO2 with L-Trp show that this part of the indole tightly
interacts with a cluster of several hydrophobic residues involving
L120, F5, Y24, Y27, and L28.39 In the case of L-Trp, the indole
H2 position is located very close to the heme.39 Similarly, 26, 36,
and 32 all exhibited a saturation transfer of around 90% for H2.
However, we observed that the less active N3-substituted
analogues 25, 35, and 31 systematically displayed a 20 to 30%
decrease in saturation transfer for H2 compared to their
regioisomers substituted in N1. Overall, these results suggest a
shift in the position of the indole core for N3-substituted
compounds, resulting in misplacement of H2. This apparent shift
of the indole position could, at least in part, explain the
difference in activity between N1- and N3-substituted com-
pounds.
Regioisomers displayed a similar binding pose, as shown in
Figure 4, for analogues 17 [IC50(enz): 15.0 μM/IC50(cell): 0.44
Figure 4. Molecular modeling of regioisomers 17 (pink) and 18
(green), bearing a trifluoroethyl from the front (a) and the side (b).
(PDB code: 5TI9)39
μM] and 18 [IC50(enz): 1.9 μM, IC50(cell): 0.19 μM] with a
particularly noticeable affinity difference. However, docking of
N1-substituted compounds resulted in a more favorable
geometry for the indole core placement. Indeed, N3-substituted
compounds display a shift of the indole core from the position
naturally adopted by the indole of L-Trp, with a shift of C2,
disturbing the interaction with the aromatic hotspots. Regarding
compounds bearing a longer sidechain with a H-bond donor
(compounds 33 to 40), our docking results suggested a possible
interaction with the propionate of the heme or with a H-
acceptor sidechain (such as Ser345) from the mobile loop. This
interaction could potentially explain the slightly better activity
observed for N3 analogues.
Phase I Metabolic Stability and Electrophilic Reac-
tivity. The potential reactivity of LM10 as a Michael acceptor
was assessed with a thiol-trapping assay using cysteamine as the
S-nucleophile and compared to that of the unsubstituted
benzotriazole 6.42−44 Following incubation with cysteamine,
the 1H NMR spectrum of LM10 quickly changed to reveal the
apparition of several new aromatic signals. In contrast, the
compound 6 spectrum stayed the same (Figure 5). Accordingly,
LC/MS analysis of LM10 ([M + H]+ = 230.1) confirmed the
apparition of a secondary product ([M + H]+ = 305.1) upon
cysteamine addition while 6 was not impacted.
Then, we evaluated the microsomal half-life of LM10 and
several representative benzotriazoles (Table 3). The recovery
percentage of LM10 after 24 h remained close to the initial
concentration. Regarding the present series, we noticed that the
nature of the N-substituent was determinant for the recovery
rate of the compounds. The presence of fluorine on the indole
did not impact the microsomal stability in compounds 8a and
37a compared to their nonfluorinated analogues. Furthermore,
in isomers 37 and 38, the localization of the N-substituent did
not impact the microsomal stability.
Interestingly, the sidechain of the different isomers always
showed a similar and low saturation transfer compared to the
aromatic protons. Therefore, the nature of the sidechains does
not appear to be useful for the interaction with hTDO2, which
opens the door to further modulation to fine-tune this family of
inhibitor physicochemical properties.
Molecular Docking. Our molecular docking studies
suggested two main hindrances for the accommodation of
different ligands in the active site of hTDO2. The first identified
hindrance was Arg144, as a shift in its sidechain was required for
the binding of the inhibitors. Displacement of this sidechain
increased with the size of the N-substituent and was more
important for N3 regioisomers. We noticed that wider Arg144
shifts correlated with lower enzymatic activity. The second
hindrance was the flexible 338−346 loop, suggesting that an
induced-fit-type binding was perhaps needed for larger
compounds. It was previously described that disordering of
this loop allows NFK to diffuse out and enables debinding of a
new L-Trp molecule.39 Its mobility was particularly noticeable
when hTDO2 was cocrystallized with an NLG919-type
analogue, widely opening the active site.28
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J. Med. Chem. 2021, 64, 10967−10980