time-dependent behavior indicative for covalent target engagement. Kinetic analysis
in a trFRET displacement assay showed the overall efficiency of covalent inactivation (described by the second-order rate constant k inact /K I ) of compound 44 (k inact /
K I ¼ 3.5 Â 10
3 M
À1 s
À1 ) to be in the same range as for pyrazinyl benzimidazole 32
from Cytopia (k inact /K I ¼ 9.0 Â 10
3 M
À1 s
À1 ), while compound 45 (k inact /K I ¼
1.7 Â 10
5 M
À1 s
À1 ) was even more efficient. Inhibitors 44 and 45 were selective for
JAK3 when tested against all JAKs in vitro and in cells, and a time-dependent
increase in inhibitory potency was observed exclusively for JAK3. Moreover, selectivity against the ten other kinases containing an equivalently positioned cysteine as
well as in a panel of 78 protein kinases was demonstrated. However, it should be
noted that compounds 44 and 32 suffered from insufficient (reversible) binding
affinity as JAK3 inhibition was outcompeted by high ATP concentrations
(IC 50 > 50 μM at 1 mM ATP; 45 was not tested). Covalent modification of
Cys909 was demonstrated by mass spectrometry for compound 45. However,
although an X-ray structure of the covalent complex between compound 32 from
Cytopia (vide supra, Fig. 8) was provided in this study, no crystal structures of
compounds 44–46 were included, and a docked model of the binding mode was
deduced instead (see the model in Fig. 13b).
In the same year, the Gray group reported a comprehensive set of covalent JAK3
inhibitors derived from WZ4002 (47), a covalent ligand of the EGFR
T790M mutant
[49, 50]. The key alteration with respect to WZ4002 was the extension of the spacer
length by replacement of the meta-substituted phenoxy linker by a benzylamine
residue (Fig. 14).
Further optimization of the other substituents and the heterocyclic hinge-binding
motif furnished a set of over 70 compounds exhaustively showing the structure–
activity relationships of this structural class. Most of the biological evaluation within
Fig. 13 (a) Covalent JAK3 inhibitors from Goedken et al. [41]. (b) Suggested binding mode
of compound 45 remodeled by covalent docking into the X-ray crystal structure with the PDB-code
4QPS using the Schrodinger Small Molecule Drug Discovery Suite
Covalent Janus Kinase 3 Inhibitors
237
in a trFRET displacement assay showed the overall efficiency of covalent inactivation (described by the second-order rate constant k inact /K I ) of compound 44 (k inact /
K I ¼ 3.5 Â 10
3 M
À1 s
À1 ) to be in the same range as for pyrazinyl benzimidazole 32
from Cytopia (k inact /K I ¼ 9.0 Â 10
3 M
À1 s
À1 ), while compound 45 (k inact /K I ¼
1.7 Â 10
5 M
À1 s
À1 ) was even more efficient. Inhibitors 44 and 45 were selective for
JAK3 when tested against all JAKs in vitro and in cells, and a time-dependent
increase in inhibitory potency was observed exclusively for JAK3. Moreover, selectivity against the ten other kinases containing an equivalently positioned cysteine as
well as in a panel of 78 protein kinases was demonstrated. However, it should be
noted that compounds 44 and 32 suffered from insufficient (reversible) binding
affinity as JAK3 inhibition was outcompeted by high ATP concentrations
(IC 50 > 50 μM at 1 mM ATP; 45 was not tested). Covalent modification of
Cys909 was demonstrated by mass spectrometry for compound 45. However,
although an X-ray structure of the covalent complex between compound 32 from
Cytopia (vide supra, Fig. 8) was provided in this study, no crystal structures of
compounds 44–46 were included, and a docked model of the binding mode was
deduced instead (see the model in Fig. 13b).
In the same year, the Gray group reported a comprehensive set of covalent JAK3
inhibitors derived from WZ4002 (47), a covalent ligand of the EGFR
T790M mutant
[49, 50]. The key alteration with respect to WZ4002 was the extension of the spacer
length by replacement of the meta-substituted phenoxy linker by a benzylamine
residue (Fig. 14).
Further optimization of the other substituents and the heterocyclic hinge-binding
motif furnished a set of over 70 compounds exhaustively showing the structure–
activity relationships of this structural class. Most of the biological evaluation within
Fig. 13 (a) Covalent JAK3 inhibitors from Goedken et al. [41]. (b) Suggested binding mode
of compound 45 remodeled by covalent docking into the X-ray crystal structure with the PDB-code
4QPS using the Schrodinger Small Molecule Drug Discovery Suite
Covalent Janus Kinase 3 Inhibitors
237