αC-helix, the floor residue Leu750, the DFG-1 residue Ile760, and Phe612 in the
P-loop (Fig. 12b).
Despite a significant impact of the lateral N-side chain structure on selectivity, it
was not possible to totally dial out affinity vs PI3Kδ in particular via chemical
modifications at this position only (compound 14 and 15 profiles in Schemes 3 and
4). Therefore, we turned our attention to the morpholine hinge binding motif
environment. There are few residues which differ in the active site of VPS34 vs
the class I PI3Ks. In particular in the hinge region, both the gatekeeper and floor
residues are mutated (Met vs Ile and Leu vs Met, respectively) offering potential
levers for selectivity [64]. The introduction of two methyl groups on the morpholine
gave rise to compound 16 exhibiting high potency on VPS34 and exquisite selectivity vs the PI3Ks and mTOR (Scheme 4).
Compound 16 was able to co-crystallize in VPS34, and a 3D structure segment is
displayed in Fig. 13a. The shape of 16 positioned in the published X-ray structure of
PI3Kδ (PDB code, 2WXL, Fig. 13b) highlighted potential steric clashes near Ile825
(GK) on one hand and with Met900 (floor residue) on the other hand.
Multi-parametric optimization eventually led to the discovery of SAR405
(Scheme 4) which affected vesicle trafficking and autophagy in cell lines and
demonstrated sustained inhibition of the autophagy process in a murine tumor
A
B
N
N
N
O
N
O
CF 3
F
F
GK Met682
C Asp644
D 743- R 744*- H 745
N-lobe Lys636
Hinge Gln683Phe684-Ile685
D 761- F 762- G 763
Cat Asn748
Phe681
Ile760
Tyr764
Gly742
Val741
Val672
GRL Phe612
GRL Leu616
Val688
Ser687
Ile634
Leu750
(floor residue)
Leu648
Fig. 12 (a) Compound 14 (carbon atoms colored in orange) in VPS34 ATP cleft, displaying a
DFG-in conformation of the protein kinase. (b) Kinase motif map of VPS34 ATP site with cpd 14.
Of note, in VPS34 as in PI3Ks, the HRD motif is inverted (DRH), the
cat
Lys is mutated in
cat
Asn,
and the αC-Glu replaced by a αC-Asp. The P-loop is poor in glycine residues therefore will not be
referred as Gly-rich loop
Compound 15
IC50s:
VPS34: 3nM
PI3Kα : 502nM
PI3Kβ : 218nM
PI3Kδ : 169nM
PI3Kγ : 7169nM
Compound 16
IC50s:
VPS34: 1nM
PI3Kα : >10µM
PI3Kβ : >10µM
PI3Kδ : >10µM
PI3Kγ : >10µM
SAR405
IC50s:
VSP34: 1nM
PI3Ks: >10µM
N
O
N
O
N
N
CF 3
S
Cl
O
N
CF 3
N
Cl
S
N
N
O
N
O
N
O
N
N
CF 3
O
Scheme 4 From hit to lead
110
L. Schio and H. Minoux
P-loop (Fig. 12b).
Despite a significant impact of the lateral N-side chain structure on selectivity, it
was not possible to totally dial out affinity vs PI3Kδ in particular via chemical
modifications at this position only (compound 14 and 15 profiles in Schemes 3 and
4). Therefore, we turned our attention to the morpholine hinge binding motif
environment. There are few residues which differ in the active site of VPS34 vs
the class I PI3Ks. In particular in the hinge region, both the gatekeeper and floor
residues are mutated (Met vs Ile and Leu vs Met, respectively) offering potential
levers for selectivity [64]. The introduction of two methyl groups on the morpholine
gave rise to compound 16 exhibiting high potency on VPS34 and exquisite selectivity vs the PI3Ks and mTOR (Scheme 4).
Compound 16 was able to co-crystallize in VPS34, and a 3D structure segment is
displayed in Fig. 13a. The shape of 16 positioned in the published X-ray structure of
PI3Kδ (PDB code, 2WXL, Fig. 13b) highlighted potential steric clashes near Ile825
(GK) on one hand and with Met900 (floor residue) on the other hand.
Multi-parametric optimization eventually led to the discovery of SAR405
(Scheme 4) which affected vesicle trafficking and autophagy in cell lines and
demonstrated sustained inhibition of the autophagy process in a murine tumor
A
B
N
N
N
O
N
O
CF 3
F
F
GK Met682
C Asp644
D 743- R 744*- H 745
N-lobe Lys636
Hinge Gln683Phe684-Ile685
D 761- F 762- G 763
Cat Asn748
Phe681
Ile760
Tyr764
Gly742
Val741
Val672
GRL Phe612
GRL Leu616
Val688
Ser687
Ile634
Leu750
(floor residue)
Leu648
Fig. 12 (a) Compound 14 (carbon atoms colored in orange) in VPS34 ATP cleft, displaying a
DFG-in conformation of the protein kinase. (b) Kinase motif map of VPS34 ATP site with cpd 14.
Of note, in VPS34 as in PI3Ks, the HRD motif is inverted (DRH), the
cat
Lys is mutated in
cat
Asn,
and the αC-Glu replaced by a αC-Asp. The P-loop is poor in glycine residues therefore will not be
referred as Gly-rich loop
Compound 15
IC50s:
VPS34: 3nM
PI3Kα : 502nM
PI3Kβ : 218nM
PI3Kδ : 169nM
PI3Kγ : 7169nM
Compound 16
IC50s:
VPS34: 1nM
PI3Kα : >10µM
PI3Kβ : >10µM
PI3Kδ : >10µM
PI3Kγ : >10µM
SAR405
IC50s:
VSP34: 1nM
PI3Ks: >10µM
N
O
N
O
N
N
CF 3
S
Cl
O
N
CF 3
N
Cl
S
N
N
O
N
O
N
O
N
N
CF 3
O
Scheme 4 From hit to lead
110
L. Schio and H. Minoux