Inhibition of 3-phosphoglycerate dehydrogenase (PHGDH) by indole amides abrogates de novo serine synthesis in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2019.07.011

Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole amides bind the NAD⁺ pocket of PHGDH. Through structure-based optimization we were able to develop compounds with low nanomolar affinities for PHGDH in an enzymatic IC₅₀ assay. In cellular assays, the most potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide series reported here represent an important improvement over previously published PHGDH inhibitors as they are markedly more potent and their mechanism of action is better defined.

Full text of DOI:10.1016/j.bmcl.2019.07.011

Accepted Manuscript
Inhibition of 3-Phosphoglycerate Dehydrogenase (PHGDH) by Indole Amides
Abrogates de novo Serine Synthesis in Cancer Cells
Edouard Mullarky, Jiayi Xu, Anita D. Robin, David J. Huggins, Andy Jennings,
Naoyoshi Noguchi, Andrea Olland, Damodharan Lakshminarasimhan, Michael
Miller, Daisuke Tomita, Mayako Michino, Taojunfeng Su, Guoan Zhang,
Andrew W. Stamford, Peter T. Meinke, Stacia Kargman, Lewis C. Cantley
PII:
S0960-894X(19)30456-1
https://doi.org/10.1016/j.bmcl.2019.07.011
BMCL 26552
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2019
2 July 2019
5 July 2019
Please cite this article as: Mullarky, E., Xu, J., Robin, A.D., Huggins, D.J., Jennings, A., Noguchi, N., Olland, A.,
Lakshminarasimhan, D., Miller, M., Tomita, D., Michino, M., Su, T., Zhang, G., Stamford, A.W., Meinke, P.T.,
Kargman, S., Cantley, L.C., Inhibition of 3-Phosphoglycerate Dehydrogenase (PHGDH) by Indole Amides
Abrogates de novo Serine Synthesis in Cancer Cells, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://
doi.org/10.1016/j.bmcl.2019.07.011
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Inhibition of 3-Phosphoglycerate Dehydrogenase (PHGDH) by Indole Amides
Abrogates de novo Serine Synthesis in Cancer Cells
Edouard Mullarkya,b, Jiayi Xuc, Anita D. Robina,b, David J. Hugginsc,d, Andy Jenningse, Naoyoshi Noguchif, Andrea
c
f
c
h
g
g
Olland , Damodharan Lakshminarasimhan , Michael Miller , Daisuke Tomita , Mayako Michino , Taojunfeng Su , Guoan
Zhangh, Andrew W. Stamfordc, Peter T. Meinkec, Stacia Kargmanc and Lewis C. Cantleya,b
a Meyer Cancer Center, Weill Cornell Medical College, New York, NY 10065
b Department of Medicine, Weill Cornell Medical College, New York, NY 10065
c Tri-institutional Therapeutics Discovery Institute, 413 East 69th Street, New York, NY 10021, United States
d Department of Physiology and Biophysics, Weill Cornell Medical College, New York, NY
e Andy Jennings Consulting, LLC
f Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd., Shonan Research Center, 26-1, Muraoka-Higashi 2-Chome, Fujisawa,
Kanagawa 251-8555, Japan
g
Xtal Biostructures, 12 Michigan Drive, Natick, MA 01760, United States
h Proteomics and Metabolomics Core Facility, Weill Cornell Medicine, New York, NY 10021
Abstract
Cancer cells reprogram their metabolism to support growth and to mitigate cellular stressors. The serine
synthesis pathway has been identified as a metabolic pathway frequently altered in cancers and there has been
considerable interest in developing pharmacological agents to target this pathway. Here, we report a series of
indole amides that inhibit human 3-phosphoglycerate dehydrogenase (PHGDH), the enzyme that catalyzes the
first committed step of the serine synthesis pathway. Using X-ray crystallography, we show that the indole
amides bind the NAD+ pocket of PHGDH. Through structure-based optimization we were able to develop
compounds with low nanomolar affinities for PHGDH in an enzymatic IC50 assay. In cellular assays, the most
potent compounds inhibited de novo serine synthesis with low micromolar to sub-micromolar activities and
these compounds successfully abrogated the proliferation of cancer cells in serine free media. The indole amide
series reported here represent an important improvement over previously published PHGDH inhibitors as they
are markedly more potent and their mechanism of action is better defined.
Keywords
PHGDH
3-phosphoglycerate dehydrogenase
Serine synthesis
Cancer metabolism
PHGDH inhibitor
inhibitor
1

Serine serves an important role in cellular metabolism. Serine is required for protein synthesis and is
incorporated into the head groups of certain lipids, such as sphingolipids and phosphatidylserine, the latter of
which is an important constituent of cellular membranes. Furthermore, serine can be metabolized to glycine,
which charges the folate pool with one carbon units. Glycine and folate one carbon units have numerous
important roles including the production of nucleotides.1,2 Serine can be imported into a cell through amino acid
transporters or can be produced de novo from glucose via the serine synthesis pathway (SSP).3,4 The SSP
consists of three sequential enzymatic reactions. The first, and rate determining, step of SSP is the conversion of
the glycolytic intermediate 3-phosphoglycerate (3-PG) to 3-phosphohydroxypyruvate (3-PHP). This reaction is
catalyzed by the NAD+ dependent enzyme 3-phosphoglycerate dehydrogenase (PHGDH). Subsequently, 3-PHP
is converted to 3-phosphoserine (3-PSER) by phosphoserine aminotransferase (PSAT) using glutamate as the
nitrogen donor. Finally, phosphoserine phosphatase (PSPH) catalyzes the magnesium dependent hydrolysis of
phosphoserine to yield serine.
Cancer cells are known to alter their metabolism not only in order to promote growth, but also to
overcome a variety of cellular stresses.5-8 The SSP has been identified as one such pathway that cancer cells
exploit for growth and stress resistance.6-13 In breast cancer and melanoma, amplifications of the gene encoding
PHGDH that lead to overexpression of the enzyme have been identified.10,11 In non-small cell lung cancer,
oncogenic mutations that activate NRF2 (nuclear factor erythroid-2-related factor) transcription factor signaling
have been reported to induce PHGDH overexpression via ATF4.13 Proliferation of cancer cell lines that
overexpress PHGDH is inhibited by shRNA mediated knockdown of PHGDH.10,11 In contrast, cancer cell lines
with low PHGDH expression are insensitive to genetic inhibition of the pathway as they rely on extracellular
serine import.10,11 Importantly, PHGDH promotes resistance to oxidative stress as the SSP maintains cellular
NADPH levels.8,13 NADPH is the major source of cellular reducing power and fuels both the glutathione and
thioredoxin systems.7 Increased PHGDH expression appears to increase the metastatic properties of cancer cells
in animal and cell culture models perhaps by mitigating the oxidative stress associated with metastasis.6,8,9,12,13
Finally, in human patients, PHGDH expression correlates with shorter survival and more aggressive disease.9-13
Together these data suggest that targeting PHGDH may be an effective therapy for treating cancers with high
SSP activity.
To date, no full-length crystal structure of human PHGDH has been reported. However, the structure of
the truncated protein, amino acids 3-314, has been reported in the PDB (entry 2G76) with both a substrate
analog, L-malate, and the NAD+ cofactor bound. The 2G76 structure shows that the truncated protein forms a
dimeric species where the PHGDH catalytic sites (1 per monomer) form at the dimerization interface of two
PHGDH monomers. Full-length PHGDH is thought to tetramerize by dimerization of two PHGDH dimers via
interactions mediated by the C-terminal domain. A catalytically inactive partial digestion fragment of PHGDH
composed of residues 93-298 that contains the NAD+ cofactor binding site missing a lid domain, which
2

partially encloses the substrate binding site was also crystalized for the purpose of fragment based drug-
screening by Unterlass et al.18
While a number of PHGDH inhibitors have been reported in the literature, identifying a potent and drug-
like PHGDH inhibitor has proved challenging. Mullarky et al. identified CBR-5884 by screening a library of
800K compounds using an in vitro PHGDH enzyme assay.14,15 CBR-5884 is a weak but selective PHGDH
inhibitor (IC50 = 33 ± 12 µM) that exhibits time dependent inhibition and disrupts the tetrameric state of
PHGDH (Fig. 1). Pacold et al. screened a 400K-compound library using a similar PHGDH enzyme assay and
discovered a thiourea class of inhibitors.16 A representative compound, NCT-503, showed moderate potency
against PHGDH (IC50 = 2.5 ± 0.6 µM) (Fig. 1). Ravez et al. identified a class of -ketothioamide derivatives of
moderate potency, inhibition constant (Ki) of 27 ± 7 µM, again using a PHGDH enzyme assay to screen a
fragment library of ~300 compounds (Fig. 1).17 While all three chemotypes showed encouraging anti-cancer
activity in cellular assays, they all suffer from weak to moderate potency. Unterlass et al. conducted differential
scanning fluorimetry (DSF) screening of a fragment library and discovered a dozen fragments that bind
PHGDH competitively to NAD+ with millimolar affinities.18 Finally, AstraZeneca scientists reported inhibitor 1
(IC50 = 0.26 µM) that was identified via a fragment-based screen and was reported to bind the adenine region of
the NAD+ pocket.19 The AstraZeneca disclosure was subsequently reviewed in Fuller et al.20 Interestingly, the
fragments reported by Unterlass et al. also seemed to bind the adenine pocket.18
Although only limited information was available for compound 1, we were interested in its sub-
micromolar binding affinity and its mechanism of action (MOA), noncovalent binding to the NAD+ pocket. We
decided to pursue 1 as a lead compound to develop more potent PHGDH inhibitors and study the biology of
inhibiting PHGDH pharmacologically in cancer cells. Over the course of our studies, multiple patent
applications from Raze Therapeutics and Boehringer Ingelheim were disclosed, all based on the indole amide
series.21-25 Here, we report the structure-activity-relationship (SAR) of analogues of 1 and our biological results.
Fig. 1. Selected small molecular PHGDH inhibitors published in the literature
In order to test the activity of 1, we developed an in vitro enzyme assay for PHGDH similar to those
previously reported with adaptations detailed in the supplementary materials.15,16 In brief, PHGDH produces
NADH from NAD+ upon oxidation of 3-PG to 3-PHP. Using diaphorase as the coupling enzyme, NADH
3

production was coupled to the reduction of resazurin to the fluorescent species resorufin. Thus, resorufin
fluorescence was measured to monitor PHGDH activity. The downstream enzymes PSAT1 and PSPH were
included in the reaction mixture to prevent product feedback inhibition of PHGDH by 3-PHP. We synthesized 1
and validated its PHGDH inhibitory activity in the enzymatic assay. Consistent with reports from surface
plasmon resonance (SPR) experiments, we obtained an IC50 of 0.238 ± 0.035 µM.26 Next, we determined the Ki
of 1 with respect to NAD+ (Fig. 2) in a manner analogous to that previously described.15,16 Compound 1 was a
mixed mode inhibitor of PHGDH with Ki = 0.214 ± 0.026 µM, as evidenced by a decrease in Vmax and
rightward shift in Km with increasing concentration of 1. These results were consistent with 1 binding the NAD+
pocket and the catalytic mechanism of PHGDH (ordered Bi Bi, NAD+ binds first).18,27 Because we were using
an enzyme coupled assay, we were concerned that 1 was actually inhibiting another enzyme in the assay system
and thus not an actual PHGDH inhibitor. However, given the agreement between the SPR data (obtained with
only PHGDH immobilized enzyme immobilized on the assay chip) and our IC50 data, and, furthermore, that 1-
mediated PHGDH inhibition was sensitive to increasing NAD+ concentration, we were confident that 1 was a
bonafide PHGDH inhibitor. The tightness of structure guided SAR data provided below further suggest that 1
inhibits PHGDH directly rather than one of the coupling enzymes.
Fig. 2. Compound 1 is a mixed mode inhibitor of PHGDH with respect to NAD+.
After confirming that compound 1 inhibited truncated PHGDH in the enzyme IC50 assay, we obtained an
X-ray crystal structure of truncated PHGDH (residues 1-313) with compound 1 bound (protein data bank entry
6PLF; supplementary materials). Similar to the previously reported X-ray crystal structures (protein data bank
entry 2G76 and 5N6C), truncated PHGDH formed a dimer.28 Compound 1 was found to bind in the adenine
binding region of the NAD+ pocket (Fig. 3). One of the key interactions was a hydrogen bond between the
amide bond N-H of 1 and the carboxylate of Asp175. The adjacent OH group also bound the carboxylate
through a hydrogen bond. The indole ring was nestled into the adenine binding region of the NAD+ pocket
4

surrounded by hydrophobic residues of P176, Y174, L151, L193, L216, T213, T207 and L210; thus, the
lipophilicity of the indole ring would contribute to inhibitor binding affinity. The carboxylate of 1 extends to the
pocket accommodating the diphosphate moiety of NAD+. This hydrophilic pocket is surrounded by two basic
guanidine sidechains from Arg155 and Arg236, which could form charge-charge interactions with the
carboxylate. The carboxylate also binds to Arg236 through a water bridge. There were several water molecules
surrounding the ligand in the structure although none are directly bridged to the protein. One water molecule
(HOH6 and HOH8 in the two chains) is buried underneath the ligand. An equivalent water molecule contacts
the phosphate of NAD+ in the PDB structure 2G76.
Fig. 3. X-ray co-crystal structure of compound 1 and PHGDH (PDB: 6PLF).
In order to interrogate the level of activity of the SSP in cells, we turned to uniformly carbon-13-labeled
glucose (13C6-glucose) tracing using LC-MS in carney cells as described previously.15 In brief, we pretreated
cells with inhibitor for 1 hour in the presence of 12C3-serine. After pretreatment, the cells were washed and
labeled with 13C6-glucose media for 2 hours still in the presence of inhibitor. From the isotopic enrichment of
serine, we could decouple newly synthesized serine from extracellular serine and serine that was synthesized
prior to tracer addition.15 Assaying serine synthesis within three hours of drug treatment was preferred to longer
term drug treatments to avoid false positives in which serine synthesis was decreased via an indirect effect, for
example, off target cell death. Concentrations of compound 1 up to 40 µM were unable to inhibit serine
synthesis as per the 13C6-glucose tracing assay (Fig. 5 A).
5

Table 1: Structure-activity relationship of 1 and its analogues
IC50
(µM)
HPLC LogD
at pH7.4
PAMPA (nm s-1
at 10 µM, pH7.4) (AB, nm s-1)
MDR1 Papp MDR1 Ratio
PSA
(Å2)
Compd.
Structure
MW
474
(BA/AB)
1
0.238
0.235
0.235
23.4
-1.86
0.88
1.67
ND
<9
<2
1
ND
150
101
80.6
150
2
3
ND
1.3
–
403
387
474
419
ND
14
–
–
–
–
6
4
0.153
209
52
1.3
80.6
63.5
80.6
93.5
5
6
24
5
1.5
2.5
3
405
437
420
–
–
ND
152 (pH 5.0)
7
8
0.037a
22
2.33
2.74
13
<1
1
9
0.030a
0.439a
0.287
2.57
2.45
2.91
<2
<2
8
ND
ND
16
433
510
462
80.6
115
10
11
<4
273
81.6
12
13
0.034a
1.58
2.40
3.03
45
ND
125
ND
451
405
80.6
63.5
154
0.63
6

14
0.37
0.99
3.10
3.10
208
252
4.9
6.1
16
17
482
482
76.4
76.4
14-en
15
16
0.015a
0.025a
2.56
2.61
<1
16
<2
ND
510
524
93.5
93.5
–
–
17
1.33
548
105
109
–
–
–
–
18
1.46
3.7
118
4.2
14
596
a measured in enzyme assay with a low PHGDH concentration described in supplemental materials; ND: not determined.
Given the high polar surface area (PSA 150 Å2) of compound 1, the presence of five hydrogen-bond
donors (HBD), and the low LogD (Table 1), we ascribed the lack of cellular activity to poor cell membrane
permeability of 1. Thus, leveraging the crystallographic information, we sought to reduce the polarity of 1 while
preserving key interactions necessary for inhibitor binding. Inspection of the co-crystal structure suggested the
indole N-H of compound 1 was not necessary for binding affinity. In addition, the water network present near
the oxazolidinone moiety was hypothesized to contribute little to the binding affinity of compound 1. Indeed,
compound 2, made by removing the oxazolidinone and masking the indole N-H with a methyl group, had the
same potency as 1, but was significantly less polar (PSA 101 Å2). Interestingly, removing the OH group (i.e. 3)
had no effect on potency, but further lowered the PSA (81 Å2) and hydrogen-bond donor count. Thus,
compound 3 had improved permeability as measured by the MDCK-MDR1 assay (A to B = 14 nm/s).
Surprisingly, the attempt to introduce a charge-charge interaction with Asp175 by replacing the OH of 2 with an
amino group as in 4 was strongly detrimental to potency. This finding could be rationalized by this amine
forming a non-binding conformation with the adjacent amide carbonyl or the introduction of a formal charged
center in the vicinity of the lipophilic surface formed by Ile177 and Ile178.
With a reduced PSA and HBD count, oxetane containing analog 5 showed moderate permeability in a
parallel artificial membrane permeability assay (PAMPA). We also made analogues linked by other groups such
7

as cyclobutane or furan. As predicted from X-ray structures, those compounds were almost as potent as the
oxetane analogues (not shown). The furan-linked compounds are chiral (adding synthetic difficulty) and the
cyclobutane-linked ones have higher LogD (poorer solubility) so oxetane 5 was preferred. In the 13C6-glucose
tracing assay, compound 5 inhibited serine synthesis in a dose dependent manner, with approximately 40%
inhibition at 10 µM and complete pathway inhibition at 40 µM (Fig. 5 B). We speculated that the dramatic
decrease in potency observed in the 13C6-glucose tracing assay relative to the enzyme IC50 assay may derive
from two factors. First, a kinetic effect where compound 5 crosses the cell membrane slowly compared to the
time frame of the tracing experiment and/or that compound 5 was highly albumin bound (plasma protein
binding >99%), greatly decreasing the free drug concentration. Experiments in which the drug pre-treatment
period was increased from one to three hours prior to initiating 13C6-glucose labeling did not increase the extent
of pathway inhibition (data not shown). Thus, the high plasma protein binding was likely a significant
contributor to the shift in potency between the enzyme and 13C6-glucose tracing assays.
The hydrogen bond between the amide N-H and the carboxylate of Asp175 was critical; replacing the
amide with an amine as in compound 6 resulted in a large loss of potency. Structural biology data suggested that
charge-charge interactions between the carboxylate of the ligand and PHGDH arginine residues may be
important. An ortho-substituting F- group next to the benzoic acid increased the potency of compound 7 by four
fold (0.037 µM). It was reasoned that the increase in potency resulted from the decrease in the pKa of the
benzoic acid. However, introducing a nitrogen heteroatom adjacent to the benzoic acid (compound 8) reduced
potency. Homologation of the benzoic acid by one methylene unit was beneficial to potency, as demonstrated
by compound 9. Unfortunately, replacing the carboxylic acid with bioisosteric groups, such as an
acylsulfonamide (compound 10) or N-methoxyamide (compound 11), significantly decreased potency.29
Introduction of an ortho-substituting F-group (compound 12) failed to enhance potency, likely because the
greater distance between the F- and the acid moiety decreases the electron-withdrawing effect.
As expected, replacing the carboxylic acid with a hydroxyl group decreased potency ten-fold (compound
13), but potency can be partially restored by the substitution with a 3-pyridyl group (compound 14). The hybrid
of compound 9 and the pyridyl group gave a more potent compound, 15; the racemate of 15 had an IC50 of
0.015 µM. We assumed that, as with compounds 14 and 14-en, one of the two enantiomers of compound 15
was more active. However, the enantiomers of 15 were not separated due to concerns around the facile
racemization of the chiral center. The increased enzymatic potency of 15 translated to greater potency in the
13C6-glucose tracing assay. Compound 15 achieved nearly 90% pathway inhibition at 10 µM (Fig. 5 C). Finally,
compound 16 showed that the carboxylate could be extended by one methylene unit and retain potency.
We obtained a co-crystal structure of rac-15 in complex with truncated PHGDH (PDB entry 6PLG;
supplementary materials). Although the resolution is moderate (3.1 Å), the structure suggested that the
carboxylate may form a bidentate H-bond with two adjacent backbone N-Hs (Ile156 and Arg155) (Fig. 4). In
8

this structure there are 8 monomers in the unit cell, all with compound 15 bound, and in some protein-ligand
systems the pyridyl may form H-bonds with the Arg236 sidechain.
Fig. 4. X-Ray co-crystal structure of compound 15 and PHGDH. (PDB entry 6PLG).
Fig. 5. Inhibition of 13C3-serine synthesis form 13C6-glucose in Carney cancer cells.
9

We profiled the selectivity of select PHGDH inhibitors (1, 4, 14, and 15) by performing biochemical
IC50 experiments on a panel of three human NAD(P)+ dependent dehydrogenases (LDHA, IDH1, MDH1)
related to PHGDH using commercially available proteins and assay kits (supplementary materials). No
significant inhibitory activity of the PHGDH inhibitors towards other dehydrogenases was observed in the
enzymatic IC50 assays at concentrations up to 10 µM (supplementary data). These data indicate that the PHGDH
inhibitors bind to the NAD+ pocket of PHGDH specifically rather being pan dehydrogenase inhibitors.
Genetic studies using RNAi suggested that greater than 75% pathway inhibition in the 13C6-glucose
tracing assay was necessary in order to achieve robust anti-cancer efficacy in cell line proliferation assays (data
not shown). To further improve potency in cell-based assays, we turned to a prodrug approach in an attempt to
overcome the low membrane permeability liability of the carboxylates. We synthesized ester prodrugs 17 and
18 which mask the negatively charged carboxylic acid moiety of 15. A study suggested that the acetoxylethyl
ester of tranexamic acid was more prone to hydrolysis in human plasma than its ethyl ester analogue (with a
half-life time at 37 oC of 6-12 min compared to 240 min).30 The potency of compound 17 in the 13C6-glucose
tracing assay was only marginally improved compared to that of the parent compound 15 (about 70% pathway
inhibition at 10 µM, Fig. 5 D). We reasoned that perhaps the intracellular hydrolysis of the ethyl ester is slow
compared to the time frame of the 13C6-glucose tracing assay and thus the improved membrane permeability of
17 was not detected in the assay as 17 did not efficiently bioconvert to its active form. Nevertheless, prodrug 18
successfully enhanced activity in the 13C6-glucose tracing assay. Compound 18 inhibited serine synthesis
approximately 40% at 0.1 µM and achieved near complete pathway inhibition at 1 µM (Fig. 5 E). The 13C-
labeling of 3-PG and pyruvate was not perturbed by drug treatment indicating that decreased serine labeling was
due to on target PHGDH inhibition rather than changes in the availability of the PHGDH substrate, 3-PG
(supplementary data).
Next, we examined whether the PHGDH inhibitors were able to inhibit the growth of a cancer cell line.
To do so, we turned to Carney cells grown in serine depleted media, as described previously.15 Carney cells can
grow in both serine replete and serine depleted medium, but removing serine from the media increases PHGDH
protein expression and renders them dependent on the serine synthesis pathway. 15 Overall, the potency of the
compounds in the 13C6-glucose tracing assay correlated with their ability to inhibit cell proliferation (Fig. 6).
Prodrug 18 was significantly more potent than its parent compound 15 and almost completely abrogated Carney
cell proliferation. Together these data establish compound 18 as a potent inhibitor of PHGDH able to abrogate
proliferation of carney cancer cells in vitro.
10

100
90
80
70
60
50
40
30
20
10
0
DMSO (0.01%)
5 (2 µM)
17 (2 µM)
15 (2 µM)
18 (2 µM)
0
24
48
72
96
120
Time (h)
Fig. 6. Growth of Carney cancer cells in serine free media treated with PHGDH inhibitors.
In summary, we have developed an indole amide series of compounds that inhibit PHGDH with low
nanomolar potencies in enzymatic assays. We were able to demonstrate that the compounds abrogated de novo
serine synthesis in cells and that they inhibited the growth of carney cancer cells in serine free medium. These
indole amide compounds represent an important improvement over previously published PHGDH inhibitors
because they are markedly more potent and have a clear mechanism of action whereby they bind the adenine
site of PHGDH in a reversible and non-covalent manner. We were able to greatly improve cellular potency by
applying prodrug approaches and achieved proof-of-concept (POC) at the cellular level. However, these ester
prodrugs are labile to hydrolysis in plasma and thus not suitable for in vivo studies. Further optimization will be
focused on developing compounds suitable for in vivo POC.
Acknowledgements
The authors gratefully acknowledge access to support that was generously provided by the Sanders Innovation
& Education Initiative, as administered by the Tri-Institutional Therapeutics Discovery Institute. LCC has
research reported in this publication that was supported by the National Cancer Institute of the National
Institutes of Health under Award Number R35CA197588. LCC has research reported in this publication that
was supported by a gift from The Roger and Susan Hertog Charitable Fund. Research reported in this
publication was supported by The Daedalus Fund For Innovation Of Weill Cornell Medicine. We would like to
thank members of the Cantley lab and the Tri-Institutional Therapeutics Discovery Institute for helpful
discussions. We thank Ce Feng Liu for discussions and help with protein purification.
Note about conflicts of interest: LCC is a founder and member of the SAB of Agios Pharmaceuticals; he is also
a co-founder, member of the SAB, and shareholder of Petra Pharmaceuticals. These companies are developing
novel therapies for cancer. LCC laboratory receives some funding support from Petra Pharmaceuticals. TDI
11

personnel, as a condition of employment, assign all ownership and decision rights related to this research
project to the originating institution WCM, MSK, or RU.
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