Please cite this article in press as: Kang et al., Discovery of Small-Molecule Selective mTORC1 Inhibitors via Direct Inhibition of Glucose Transporters,
Figure 3. NV-5440 and Its Analogs Block
A
Glucose Uptake via GLUTs
(A) NV-5440 inhibits mTORC1 in the presence and
absence of glucose. On starvation of glucose or
amino acids for 1 h, MCF7 cells were treated with
increasing concentrations of NV-5440 for 1 h and
then restimulated by glucose or amino acids for
15 min. Resulting lysates analyzed by immunoblot-
ting for the indicated proteins and phosphorylation
states.
C
B
(B) NV-5440 inhibits mTORC1 by blocking glucose
uptake with an IC50 of 36 nM. Radioactively labeled
[3H]2-DG uptake assay following 20 min compound
treatment in MCF7 cells.
(C) Cell-based pS6K1 AlphaLISA assay potency
versus radioactive glucose uptake assay IC50 for 129
compounds in the NV-5440 series in MCF7 cells
(all data shown as pIC50, r = 0.835).
(D and E) NV-5440 displays strong selectivity for
GLUT1 over GLUT2, 3, 4, and 5. CHO-K1 cells were
stably transfected with rat GLUT1, GLUT2, or GLUT3
(see graph D), as were HEK-293T cells with human
GLUT4 or GLUT5 (see graph E). The transfected cells,
except for GLUT4, were incubated with an appropriate
glucose concentration, or fructose concentration with
or without NV-5440 and 1 mM rotenone. The CellTiter-
Glo Luminescent Cell Viability Assay was then used to
measure ATP levels. Assay was normalized to the
control cytochalasin B. Activity on GLUT4 was
measured with the Glucose Uptake-Glo Assay.
D
E
that the GLUT1 binding activity of the compounds inhibits incorporation of [3H]2-DG into cells was detected by scintillation
S6K1 phosphorylation in cells.
counting of cell lysates. This assay demonstrated that NV-5440
rapidly and potently inhibited glucose transport into cells in a
dose-dependent manner. Full inhibition of glucose uptake by
NV-5440 was achieved at a concentration of 1 mM and above
(Figure 3B). The IC50 for glucose uptake inhibition was 36 and
140 nM in MCF7 and HUVECs, respectively. NV-5440 can inhibit
glucose uptake in as little as 10 min in MCF7 cells (Figure S6), the
shortest time point tested, suggesting that glucose uptake inhi-
bition occurred rapidly through inhibition of a direct uptake
mechanism. A number of structurally related compounds were
also tested in the glucose uptake assay, and the resulting IC50
values correlated closely with the inhibition of S6K1 phosphory-
lation (r = 0.913, Bravais-Pearson), indicating that there is a
correlation between glucose uptake and mTORC1 inhibition
NV-5440 Perturbs the Glucose-Sensing Mechanism of
mTORC1
To verify that NV-5440 acts via the glucose-sensing mechanism
to inhibit mTORC1, we assessed the impact of NV-5440 under
different nutrient starvation conditions in MCF7 cells. Under
glucose-stimulated conditions, NV-5440 inhibited mTORC1 in a
dose-dependent manner. However, when the cells were treated
with NV-5440 under glucose-starved conditions, the maximal in-
hibition of mTORC1 at 10 mM did not exceed the level of inhibition
achieved by glucose starvation alone (Figure 3A). The data
suggest that NV-5440 and glucose withdrawal inhibit mTORC1
via an overlapping mechanism, consistent with NV-5440 target-
ing the glucose transport function of GLUT1. Conversely, when
cells were treated with NV-5440 under amino acid-starved
conditions, NV-5440 was able to inhibit mTORC1 beyond the
baseline achieved by amino acid starvation alone (Figure 3A).
Therefore, we conclude that NV-5440 works independently of
the amino acid-sensing arm of the mTORC1 pathway.
GLUT Specificity Testing
Glucose transporters belong to a large family of structurally
similar transporter proteins that are separated into three classes.
Class 1 comprises the well-characterized GLUTs 1 through 4,
while sequence homology has identified additional GLUTs, char-
acterized as classes 2 and 3. Therefore, we attempted to mea-
NV-5440 Potently Inhibits Glucose Uptake in Cells
To confirm functional inhibition of the GLUT1 transporter, we sure not only the overall impact on glucose uptake but also the
tested the impact of NV-5440 on glucose uptake. Glucose trans- specificity of NV-5440 and its analogs for the various transporter
port was assessed by measuring the uptake of a labeled glucose classes. CHO-K1 cells were stably transfected to overexpress
analog, 2-deoxy-D-[3H]glucose ([3H]2-DG), in MCF7 and primary rat GLUT1, GLUT2, or GLUT3, and HEK-293T cells were trans-
human umbilical vein endothelial cells (HUVECs). In brief, cells fected to overexpress human GLUT4 or GLUT5. The cells were
were incubated in glucose-free medium to deplete intracellular treated with the oxidative phosphorylation inhibitor rotenone
glucose, exposed to compounds and [3H]2-DG for 20 min. The to ensure that any contribution of mitochondrial activity was
6
Cell Chemical Biology 26, 1–11, September 19, 2019