Methylation of the phenyl ring was tolerated by the PI3Kβ binding site only in
position 4 (indoline 25a, Fig. 17b), but no improvement of solubility was noticed.
Methylation of the pyrimidinone core (indoline 28) was tolerated regarding activity,
slightly increased solubility compared to 24, but this avenue was dismissed since it
introduced a metabolic hot spot. Methylation in position 3 had no effect in contrary
to position 2 which brought an outstanding improvement of solubility. The racemic
indoline 27 was separated into its two pure enantiomers, and another unexpected
finding was obtained: the (R)-isomer-27 was found equipotent on PI3Kβ and PI3Kδ,
whereas the (S)-isomer-27 retained selectivity vs PI3Kδ but at the expense of
activity on PI3Kβ (Table 2).
Whereas PI3Kβ had been refractory to intensive efforts deployed in house and
elsewhere to find conditions and constructs in order to obtain crystals of the kinase
domain [74], we finally succeeded and reported for the first time the X-ray structure
solved at 2.8 Å of a propeller shaped ligand ((S)-27) in complex with p110β (http://
www.rcsb.org/structure/4BFR). Moreover (S)-27 could be also co-crystalized in
PI3Kδ (2.6 Å), and we realized that both co-structures were extremely similar
(Fig. 18) despite one log difference in biochemical activity exhibited by (S)-27
(Table 2) [79].
The two co-structures obtained were otherwise very consistent with those previously collected in the pyrimidinone series using PI3Kγ as a surrogate protein (e.g.,
Fig. 16a) and for other publicly reported propeller-shaped selective ligands
[80]. With respect to PI3Kβ, key and common observations were interaction of the
morpholine moiety with the hinge region via the main-chain nitrogen of Val848,
movements of the P-loop Trp781 and Met773 opening a specific pocket where the
indoline moiety stacks.
From these findings emerged the following paradoxes:
1. A similar conformational change can occur across the different PI3K isoforms to
create a specific pocket.
2. Biochemically inactive but propeller-shaped molecules can co-crystallize in
PI3Ks and occupy the so-called selectivity pocket.
Table 2 Biochemical activities of compounds (R)-27 and (S)-27 on five kinases, in nM
Compound Structure
PI3Kα
(IC 50 )
PI3Kβ
(IC 50 )
PI3Kδ
(IC 50 )
PI3Kγ
(IC 50 )
pAkt inh. PC3
(IC 50 )
(S)-27
N
O
N
N
H
O
O
N
1,000
23
468
10,000
49
(R)-27
N
O
N
N
H
O
O
N
569
6
6
3,315
12
116
L. Schio and H. Minoux
position 4 (indoline 25a, Fig. 17b), but no improvement of solubility was noticed.
Methylation of the pyrimidinone core (indoline 28) was tolerated regarding activity,
slightly increased solubility compared to 24, but this avenue was dismissed since it
introduced a metabolic hot spot. Methylation in position 3 had no effect in contrary
to position 2 which brought an outstanding improvement of solubility. The racemic
indoline 27 was separated into its two pure enantiomers, and another unexpected
finding was obtained: the (R)-isomer-27 was found equipotent on PI3Kβ and PI3Kδ,
whereas the (S)-isomer-27 retained selectivity vs PI3Kδ but at the expense of
activity on PI3Kβ (Table 2).
Whereas PI3Kβ had been refractory to intensive efforts deployed in house and
elsewhere to find conditions and constructs in order to obtain crystals of the kinase
domain [74], we finally succeeded and reported for the first time the X-ray structure
solved at 2.8 Å of a propeller shaped ligand ((S)-27) in complex with p110β (http://
www.rcsb.org/structure/4BFR). Moreover (S)-27 could be also co-crystalized in
PI3Kδ (2.6 Å), and we realized that both co-structures were extremely similar
(Fig. 18) despite one log difference in biochemical activity exhibited by (S)-27
(Table 2) [79].
The two co-structures obtained were otherwise very consistent with those previously collected in the pyrimidinone series using PI3Kγ as a surrogate protein (e.g.,
Fig. 16a) and for other publicly reported propeller-shaped selective ligands
[80]. With respect to PI3Kβ, key and common observations were interaction of the
morpholine moiety with the hinge region via the main-chain nitrogen of Val848,
movements of the P-loop Trp781 and Met773 opening a specific pocket where the
indoline moiety stacks.
From these findings emerged the following paradoxes:
1. A similar conformational change can occur across the different PI3K isoforms to
create a specific pocket.
2. Biochemically inactive but propeller-shaped molecules can co-crystallize in
PI3Ks and occupy the so-called selectivity pocket.
Table 2 Biochemical activities of compounds (R)-27 and (S)-27 on five kinases, in nM
Compound Structure
PI3Kα
(IC 50 )
PI3Kβ
(IC 50 )
PI3Kδ
(IC 50 )
PI3Kγ
(IC 50 )
pAkt inh. PC3
(IC 50 )
(S)-27
N
O
N
N
H
O
O
N
1,000
23
468
10,000
49
(R)-27
N
O
N
N
H
O
O
N
569
6
6
3,315
12
116
L. Schio and H. Minoux