Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.02.016

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux throug

Full text of DOI:10.1016/j.bmc.2018.02.016

Accepted Manuscript
Discovery and optimization of piperazine-1-thiourea-based human phospho-
glycerate dehydrogenase inhibitors
Jason M. Rohde, Kyle R. Brimacombe, Li Liu, Michael E. Pacold, Adam
Yasgar, Dorian M. Cheff, Tobie D. Lee, Ganesha Rai, Bolormaa Baljinnyam,
Zhuyin Li, Anton Simeonov, Matthew D. Hall, Min Shen, David M. Sabatini,
Matthew B. Boxer
PII:
S0968-0896(18)30042-7
https://doi.org/10.1016/j.bmc.2018.02.016
BMC 14202
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 January 2018
6 February 2018
13 February 2018
Please cite this article as: Rohde, J.M., Brimacombe, K.R., Liu, L., Pacold, M.E., Yasgar, A., Cheff, D.M., Lee,
T.D., Rai, G., Baljinnyam, B., Li, Z., Simeonov, A., Hall, M.D., Shen, M., Sabatini, D.M., Boxer, M.B., Discovery
and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors, Bioorganic &
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate
dehydrogenase inhibitors
Jason M. Rohdea*, Kyle R. Brimacombea, Li Liua, Michael E. Pacoldb-h, Adam Yasgara, Dorian
M. Cheffa, Tobie D. Leea, Ganesha Raia, Bolormaa Baljinnyama, Zhuyin Lia,i, Anton Simeonova,
Matthew D. Halla, Min Shena, David M. Sabatinib-e, Matthew B. Boxera,j
a
National Center for Advancing Translational Sciences, National Institutes of Health, 9800
Medical Center Drive, Rockville, MD, 20850, USA
b Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142,
USA
c Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
d Koch Institute for Integrative Cancer Research, 77 Massachusetts Avenue, Cambridge, MA
02139, USA
e Broad Institute of Harvard and Massachusetts Institute of Technology, 7 Cambridge Center,
Cambridge, MA 02142, USA
f Dana-Farber Cancer Institute, Longwood Center, 350 Longwood Avenue, Boston, MA 02215,
USA
g Department of Radiation Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue,
Boston, MA 02215, USA
h Current address: Department of Radiation Oncology, New York University Medical Center,
522 First Avenue, Smilow 907, New York, NY 10016
i Current address: Bristol-Myers Squibb, Lead Discovery and Optimization, 3551 Lawrenceville
Road, Princeton, NJ 08540
j Current address: Nexus Discovery Advisors, 7820B Wormans Mill Road, Suite 208, Frederick,
MD 21701
* To whom correspondence should be addressed:
Jason Rohde, Ph.D.
9800 Medical Center Dr.
Rockville, MD 20850
Phone: 301-827-1755. Fax: 301-217-5736. Email: jason.rohde@nih.gov

Keywords:
PHGDH
inhibitor
serine
Abstract
Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism
of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose,
phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for
this important amino acid and represents a putative target in oncology. To discover inhibitors of
PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput
screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling
PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked
improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin
system enabled a red-shifted detection readout, minimizing interference due to compound
autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH
inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified.
Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-
503) were identified. These molecules demonstrated improved target activity and encouraging
ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its
role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay
development and medicinal chemistry leading to the development of NCT-502 and -503 reported
in Nat. Chem. Biol. 2016, 12 (6), 452-8.
Page 2 of 33

Introduction
Serine is an important biochemical building block, being incorporated wholly into proteins as
well as into DNA and RNA bases as one-carbon units or as serine-derived glycine. Given this
crucial role, proliferating cells obtain serine exogenously or derive it from glucose. By catalyzing
the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate
dehydrogenase (PHGDH) controls flux through this biosynthetic pathway. A series of PHGDH-
overexpressing breast cancer cell lines display a dependence on this pathway as genetic
suppression of PHGDH was shown to be toxic even in the presence of exogenous serine.1-2
Given PHGDH’s potential as a drug target in certain cancers, a number of PHGDH inhibitors
have been reported.3-11 AstraZeneca used a fragment-based lead discovery approach followed by
medicinal chemistry optimization to develop an indole derivative (Figure 1) with sub-
micromolar KD.4-5 This approach outperformed high-throughput screening (HTS) hits to deliver
the most potent inhibitor reported to date. Cantley and colleagues used a biochemical HTS to
identify a moderately potent PHGDH inhibitor (CBR-5884, Figure 1) with an IC50 of ~33 M,
which displays in vitro activities suggestive of a covalent, allosteric modulator.6 Additionally,
the Cantley lab, in conjunction with the Feron and Frédérick groups, through the convergence of
pharmacophores developed another covalent inhibitor, namely an -ketothioamide, which
displayed encouraging cellular activity.11 A patent filed by Raze Therapeutics disclosed a novel
chemotype (representative example shown in Fig 1), but no detailed information on inhibitory
activity was shown.9 Further, a computational approach led to the discovery of a pair of allosteric
inhibitors of PHGDH (PKUMDL-WQ-2101, PKUMDL-WQ-2201) and despite modest
potencies they were shown to be efficacious in vivo.10 Overall, the community has developed a
useful and diverse set of PHGDH modulators
Page 3 of 33

Figure 1. Reported PHGDH inhibitors
We have previously reported reversible inhibitors of PHGDH (NCT-502, NCT 503, Figure 1)
with low-micromolar biochemical inhibition that demonstrated inhibition of PHGDH-dependent
cancer cell growth, and reduced glucose-derived serine production in cells.8 Activity was also
demonstrated in orthotopic xenograft tumors.
Here, we describe the development and optimization of a high-throughput amenable PHGDH
biochemical activity assay to enable screening in 1536-well format. This assay was utilized to
conduct a quantitative high-throughput screen (qHTS) to uncover small molecule inhibitors of
PHGDH for further development. In addition, we describe the optimization of a thiourea
chemotype into a set of in vitro and in vivo probes to study PHGDH biology.
Results
The optimized PHGDH assay used for the HTS utilized a coupled enzyme system to provide
robust recombinant PHGDH activity and to shift detection of dehydrogenase activity from a
classic NADH-based blue fluorescent readout to a red-shifted resorufin-based readout. This
coupled biochemical assay system includes two human enzymes – phosphoserine transaminase
(PSAT1) and phosphoserine phosphatase (PSPH) – which naturally participate in the serine
biosynthetic pathway immediately downstream of PHGDH, along with a bacterial enzyme,
diaphorase from Clostridium kluyveri, to couple NADH levels to the resazurin/resorufin
fluorescent dye system (Figure 2a).12
Page 4 of 33

a)
b)
c)
d)
e)
f)
g)
h)
Page 5 of 33

Figure 2. PHGDH assay development. a) Scheme of PHGDH enzymatic reaction coupled to
diaphorase for fluorescence detection. b) The diaphorase assay was run using a resazurin titration
to determine the substrate’s Km (0.05 mM), and feedback inhibition was observed at substrate
concentrations above 0.25 mM. Data are average of 48 wells per condition, and error bars
represent s.d. c) PHGDH activity (10 nM) was measured utilizing varying stoichiometries of
PSAT1 and PSPH to relative contributions to PHGDH assay turnover. Data represent the change
in fluorescence (∆RFU) over 30 min (48 wells/condition), and error bars represent s.d.
Conditions for the coupled PHGDH assay were determined by testing titrations of d) diaphorase,
e) 3PG, f) NAD+ and g) glutamate to identify ideal assay parameters (i.e. Kms for the substrates
3PG and NAD+, and non-limiting concentrations for the coupling reagents diaphorase and
glutamate). For all studies, data are average of 48 wells per condition, and error bars represent
s.d. h) A time course demonstrating robust, near-linear kinetics is shown for the fully-coupled
assay in the presence and absence of diaphorase. Data are average of 48 wells per condition, and
error bars represent s.d.
Assay Development & Optimization
The coupling efforts detailed here were undertaken after initial adaptation of a PHGDH assay
containing solely the dehydrogenase and its substrates, which quantified NADH production by
directly measuring NADH fluorescence, yielded low enzymatic turnover and required a lengthy
reaction period to achieve appreciable signal (data not shown). Furthermore, given the
prevalence of blue autofluorescence among small molecules in screening collections, we
anticipated a need to shift the primary assay away from the blue NADH-based fluorescent
readout.13
To address these issues, we coupled NADH production to diaphorase, which has been utilized in
other dehydrogenase and NAD+/NADH systems to introduce a separate, red-shifted detection
Page 6 of 33

dye, resazurin.14 Diaphorase is an oxidoreductase enzyme that can utilize NADH or NADPH to
catalyze the reduction of resazurin to fluorescent resorufin. Diaphorase-coupling provides a two-
fold benefit in the case of PHGDH by 1) generating resorufin, a robust detection dye with HTS-
amenable red-shifted fluorescence, with 1:1 stoichiometry to NADH, and by 2) regenerating
NAD+ and preventing accumulation of NADH by the primary PHGDH reaction (which can slow
enzyme kinetics). We tested Clostridium kluyveri diaphorase for this purpose by determining
optimal resazurin and diaphorase concentrations using PHGDH assay buffer and substrate
conditions. Titration of resazurin using 0.5 mM NADH and 0.015 mg/mL diaphorase yielded a
Km of 50 M for resazurin, and determined that maximal production of resorufin signal occurred
at 250 M substrate; concentrations above this threshold were found to demonstrate feedback
inhibition with reduced signal (Figure 2b). For coupling purposes, resazurin was included at 0.1
mM, which was high enough to achieve a non-limiting concentration relative to NAD+/NADH in
the enzyme system, yet low enough to avoid any potential feedback inhibition.
PHGDH has been shown to be susceptible to feedback inhibition15 by its product
phosphohydroxypyruvate (p-Pyr), so the assay system was further coupled to two downstream
enzymes, PSAT1 and PSPH, to minimize p-Pyr accumulation and help ‘drive’ the reaction
forward. Optimal stoichiometry of PHGDH, PSAT1 and PSPH were determined by titrating the
downstream enzymes PSAT1 and PSPH in the presence of 10 nM PHGDH and non-limiting
substrate concentrations (1 mM NAD+, 2 mM 3-phosphoglycerate [3PG], 0.625 mM glutamate),
using NADH fluorescence as a direct readout of biochemical reaction progression. Inclusion of
PSAT1 and PSPH led to increased PHGDH turnover and greater assay signal at all tested
concentrations and stoichiometries, though PSAT1 concentrations were found to most strongly
influence overall assay signal (Figure 2c). Inclusion of 500 nM PSAT1 and 500 nM PSPH
yielded a 36% increase in signal over the lowest concentrations tested (100 nM each); though
higher PSAT1 and PSPH concentrations did provide further increases in signal. Therefore, 500
nM concentrations of both enzymes were chosen to balance assay signal with protein
requirements.
Next, we performed a diaphorase titration in the presence of either PHGDH alone, PHGDH with
PSAT1 and PSPH (positive control), or PSAT1 and PSPH alone (no PHGDH, negative control).
The fully-coupled PHGDH/PSAT1/PSPH/diaphorase assay conditions yielded significantly
Page 7 of 33

greater assay signal and turnover than other conditions, with 2-7-fold increased turnover
compared to PHGDH/diaphorase in the absence of PSAT1 and PSPH (Figure 2d), confirming
that the coupled assay increases PHGDH enzymatic activity. Though maximal assay signal was
seen at 0.025 mg/mL diaphorase in the fully-coupled conditions, diaphorase was included in
subsequent screening at a higher concentration (0.1 mg/mL) to buffer against any false positive
readings in the event of direct inhibition of diaphorase itself.
With coupling conditions determined, the assay system substrates 3PG, NAD+ (PHGDH) and
glutamate (PSAT1) were individually titrated to determine Km values and saturating
concentrations. Km values for 3PG and NAD+ were determined to be 50 M and 150 M,
respectively, and these concentrations were adopted into the final assay conditions to facilitate
detection of small molecule competitors of either substrate (Figures 2e, f). Glutamate was found
to be non-limiting above 310 M, so 625 M glutamate was utilized in the final assay to ensure
saturation for the PSAT1/PSPH coupling reaction (Figure 2g). The final, fully-coupled PHGDH
assay demonstrated robust, linear turnover over 20 min, suggesting it was amenable to screening
(Figure 2h). These conditions represent the final coupled PHGDH assay utilized to conduct all
subsequent screening and validation work (Table 1).
Table 1. Primary PHGDH coupled assay protocol
Step
1
Value
3 µL
23 nL
Description
Substrate mix in assay buffer
Dilution series (4-11 point series in DMSO)
Reagent Addition
Compound Addition
2
3
4
Reagent Addition
Detection
1 µL
PHGDH enzyme mix in assay buffer
ViewLux initial read, T0 min
Fluorescence
5
6
Incubation
Detection
20 min
Fluorescence
Room temperature incubation
ViewLux final read, T20 min
Notes
Substrate buffer: 50 mM triethanolamine, 10 mM MgCl2, 0.05% BSA, 0.01% Tween20, 10 µM
EDTA, 0.625 mM glutamate, 0.05 mM 3-phosphoglycerate, 0.1 mM resazurin, 500 nM PSAT1,
500 nM PSPH and 0.1 mg/mL diaphorase
1
2
3
Pintool transfer
PHGDH enzyme buffer: 50 mM TEA, 10 mM MgCl2, 0.05% BSA, 0.01% Tween20, 0.15 mM
NAD+, 10 nM PHGDH
4
5
6
ViewLux fluorescence read (ex525/em598, 5 sec exposure, 5000 excitation energy)
Room temperature
ViewLux fluorescence read (ex525/em598, 5 sec exposure, 5000 excitation energy)
Page 8 of 33

Following initial publication of the discovery and characterization of NCT-502 and NCT-503, it
was reported that the prescence of an N-His epitope led to structural alterations in PHGDH, and
that N-His PHGDH was found to have a significantly lower specific activity than untagged
PHGDH.16 It is worth noting that the assay optimization and screening reported here was
conducted using N-His-tagged PHGDH, so adaptation of this protocol to utilize untagged
PHGDH may require additional optimization to account for potential differences in specific
activity.
Pilot Screening
To compare the performance of the optimized assay to the original uncoupled version,
preliminary screening was conducted using the LOPAC®1280 library. In this pilot screen, the
uncoupled NADH assay resulted in interference from compound fluorescence, which was of a
considerably greater magnitude than the NADH fluorescence resulting from PHGDH turnover
and NADH production (Figure 3a).
a)
Page 9 of 33

b)
c)
d)
Figure 3. PHGDH preliminary screening and optimization. a) Plate images are shown for
T=0 (left plates) and T=30 min (right plates) reads from two PHGDH screens against the
LOPAC library (11.5 M). The two top plates demonstrate the autofluorescence of LOPAC
library compounds in the PHGDH assay utilizing NADH-based detection, while the bottom two
plates show well signal from the PHGDH assay using resazurin-based detection. Very few
autofluorescent compounds can be observed in the resazurin wavelength, providing a clearer
landscape for observing potential PHGDH inhibition. b) Examples are shown of false-positive
hits identified from LOPAC in the PHGDH assay utilizing NADH-based detection. All
corresponding compounds were completely inactive in the diaphorase/resazurin-based PHGDH
assay, demonstrating an advantage of migrating to the 598 nm detection realm. Assay signal
windows observed during LOPAC screens using c) NADH-linked or d) resorufin-based readouts.
Data are average of 64 wells per condition, and error bars represent s.d.
Using ∆30 min data at 57 µM compound, where inhibition ≥50% was defined as active, the
NADH-fluorescence assay yielded 103 hits out of 1280 compounds (hit rate of 8%). In contrast,
the PSAT1/PSPH/diaphorase-coupled assay showed nearly 3-fold fewer autofluorescent
Page 10 of 33

compound artifacts, (37 out of 1280 compounds, hit rate of 3%) and at a comparatively lower
magnitude relative to resorufin signal.
During these screens, plates were measured every 10-15 min for a total of 30 min, and 32
positive control (10 nM PHGDH enzyme) and negative control (no PHGDH enzyme) wells were
included on each 1536-well plate to monitor assay performance. The uncoupled assay (direct
NADH detection) yielded a ∆RFU of only 20 after 30 min (Figure 3c), while the diaphorase
assay demonstrated a ∆RFU of nearly 1000, representing a 50-fold increase in signal over the
same time period (Figure 3d). The coupled assay also demonstrated superior performance to the
original assay, for both signal-to-background (5.1 vs 3.7) and Z’ (0.85 vs 0.80).
Primary Quantitative High-throughput Screening
The coupled PHGDH assay was utilized to screen our in-house collection of more than 400,000
small molecules. In this qHTS screen, the inhibition associated with each well was computed
from the endpoint and normalized against control wells. The percent inhibition at each of the
concentrations of inhibitor tested was fit to a sigmoidal dose-response curve (Hill equation)
using in-house-developed software (https://tripod.nih.gov/curvefit/) to determine the compound
IC50 values. Across >500 1536-well plates, assay performance was strong with Z’ generally >0.7
and S:B >20 (Figure 4a). Analysis of these dose-response curves resulted in 1342 high-quality
actives that showed strong inhibition with curve classes 1-3, efficacy over 50% and IC50 ≤ 20
µM (Figure 4b). These hit compounds were then thoroughly evaluated for both promiscuity and
synthetic tractability to eliminate compounds that did not offer good starting points for lead
optimization. A total of 239 prioritized inhibitors were reacquired and screened in 11-point dose-
response in the primary screening assay to confirm their activity.
a)
b)
Page 11 of 33

c)
d)
e)
Figure 4. PHGDH high-throughput screen results. a) Assay performance statistics (Z-factor,
top, and signal-to-background ratio, bottom) are shown for each plate across the entire PHGDH
qHTS campaign. b) A breakdown of the number and quality of qHTS hits is shown. High
confidence actives are those demonstrating curve classes of -1.1, -1.2 and -2.1, while low
confidence actives demonstrated curve classes of -1.3, -1.4, -2.2 or -3. c) The PHGDH assay hit
triage funnel, showing the assays used to winnow the initial collection of screening hits, along
with the number of compounds remaining after each stage. d) Dose-response data showing
activity of screening hit (1) in the assay hit triage funnel. Data is shown for 1 in the primary
coupled screen and cherry-pick validation (PHGDH, PSAT1, PSPH and diaphorase), the
uncoupled secondary assay (PHGDH and diaphorase), the orthogonal secondary assay (PHGDH,
PSAT1 and PSPH), and the diaphorase-only counterscreen assay. e) Dose-response data
demonstrating selectivity of the screening hit 1 against a panel of dehydrogenase assays
(PHGDH, IDH1, LDHA and GAPDH). Data are average of 3 experiments, and error bars
represent s.d.
Page 12 of 33

Hit Validation and Triage
A number of secondary assays and counterscreens were selected and utilized to refine and triage
initial screening hits (assay hit triage funnel shown in Figure 4c). This triage panel included 1)
validation in the primary assay using expanded 11-point dose-response, 2) triage of PAINS17-18
or other chemotypes/scaffolds not amenable to medicinal chemistry optimization, 3) orthogonal
validation in the original uncoupled PHGDH assay (PHGDH alone, using NADH fluorescence),
4) counterscreens against diaphorase alone, 5) orthogonal screening against the coupled
PHGDH/PSAT1/PSPH reaction using NADH fluorescence (without diaphorase), and 6)
counterscreens against unrelated dehydrogenases (isocitrate dehydrogenase [IDH1], lactate
dehydrogenase A [LDHA] and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]).
The activity of 1 (for the chemical structure of compound 1 see Table 2) seewas subsequently
characterized in dose-response using the outlined assay triage hit funnel (Figure 4d). The
primary screen and secondary cherry-pick validation activity of 1 was found to be in close
agreement (14.1 M vs 15.3 M). In the uncoupled PHGDH assay, which utilized diaphorase
and resazurin for detection without PSAT1 and PSPH, 1 displayed an increase in potency (5.4
M), suggesting that its activity was independent of the presence/activity of the downstream
enzymes. Similarly, 1 retained activity in the orthogonal PHGDH assay (17.2 M), which
included PSAT1 and PSPH but utilized NADH fluorescence as a direct measure of turnover,
demonstrating that its activity was not dependent on the diaphorase/resazurin coupling system.
This observation was further confirmed in the diaphorase counterscreen assay, which showed no
appreciable inhibition of diaphorase (<9.3% inhibition at the top concentration of 57.5 M).
Taken together, the assay panel suggested that 1’s activity was directly dependent on PHGDH,
rather than any of the coupling enzymes.
Select hits were advanced for selectivity testing, which included a small panel of dehydrogenases
(namely IDH1, LDHA and GAPDH) to exclude pan-dehydrogenase inhibitors. A number of hit
compounds were found to demonstrate strong selectivity toward PHGDH over other
dehydrogenases, with little comparative off-target activity. Notably, the screening hit 1 displayed
complete selectivity toward PHGDH, with no discernable off-target dehydrogenase inhibition
(Figure 4e).
Medicinal Chemistry & SAR Exploration
Page 13 of 33

Based on this panel of assays, a series of 2-pyridinyl-N-(4-aryl)piperazine-1-carbothioamides
emerged as the top, validated chemotype, as low micromolar inhibitors of PHGDH. This
chemotype was explored earlier during another NCGC structure-activity campaign to develop
inhibitors of bacterial phosphopantetheinyl transferase (PPTase)19. Utilizing compounds from
this previous medicinal chemistry effort, we were able to rapidly establish preliminary structure-
activity relationships (SAR) beyond hit 1 (Table 2).
Table 2. Initial SAR focusing on the thiourea group of hit 1
IC50(M)a
Cpd
R
% inhibition
92
15.3
1
ND
ND
ND
38 (max)
16 (max)
2 (max)
2
3
4
ND
ND
37 (max)
9 (max)
5
6
Page 14 of 33

21
45
7
aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay. ND = not determined; for compounds with % inhibition at 57 M
weaker than 40%, IC50 was ND.
We began our PHGDH SAR investigations with a focus on the necessity of the thiourea group
(Table 2). This feature of the pharmacophore proved essential to activity as the urea (3),
guanidine (4), and thioamide (5) replacements led to significant loss of PHGDH activity. Despite
the potential for reactivity posed by the presence of a thiocarbonyl, this class of molecules has
previously been shown to reversibly inhibit PHGDH in vitro.8 Additionally, other minor
modifications to this region of the molecule were not tolerated such as N-methylation (2),
shortening of the linkage from the thiocarbonyl to the pyridine (6), or lengthening the linkage
(7).
Subsequently, we shifted our SAR analysis to the aminopyridine portion of the chemotype
(Table 3). The pyridine in this region was required to maintain target inhibition as phenyl
variant 8 lost all potency. The positioning of the pyridine nitrogen also proved to be optimal at
the two-position as the 3- and 4-pyridyl analogs 10 and 11 lost both potency and efficacy in
comparison to compound 9. A second nitrogen proximal to the thiourea nitrogen in the form of a
pyrimidine (12) led similarly to diminished PHGDH activity as did substitution of the pyridine
with electron-withdrawing groups (analogs 13 and 14). The addition of a second methyl group
resulted in an improvement in potency, whereas expansion of the pyridine ring system in the
form of a quinoline (analogs 16 and 17) showed small perturbations in target activity.
Table 3. Brief SAR of the aminopyridine portion of hit 1
IC50(M)a
Cpd
R
% inhibition
Page 15 of 33

15.3
ND
92
1
8
20 (max)
6.8
8.9
14
93
67
55
9
10
11
37
6.4
13
111
61
12
13
14
73
6.3
10
21
92
90
15
16
17
119
aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay. ND = not determined; for compounds with % inhibition at 57 M
weaker than 40%, IC50 was ND.
A key feature of our approach to the optimization of this chemotype involved the evaluation of
structure-property relationships (SPR) in parallel to all of our SAR work. We performed a set of
in vitro absorption, distribution, metabolism and excretion (ADME) assays on all compounds
including aqueous kinetic solubility, rat liver microsomal stability (single point), and parallel
artificial membrane permeability assay (PAMPA). At this stage of our investigations, we
leveraged improved SPR observations as compound 15 displayed improved microsomal stability
(rat liver microsomes t1/2 = >30 min) compared to analog 9 (t1/2 = 16 min). Thus,
dimethylpyridine was carried forward for subsequent SAR studies on the remaining regions of
the chemotype. Furthermore, although only a small sample of analogs probing this section of the
hit molecule 1 are displayed here, a broader exploration carried out later in the SAR campaign
Page 16 of 33

confirmed, in the same way, that this chemotype would not accommodate alterations to the
pyridyl ring system.
Having established that the 2-pyridine and the thiourea are important features of the
pharmacophore, we shifted our attention to the arene affixed to the piperazine in order to assess
the flexibility therein (Table 4). Movement of the trifluoromethyl group around the arene proved
to be well-tolerated with the best location being para to the piperazine (19). Unsubstituted
pyridines lost most of their PHGDH activity regardless of their positioning (analogs 20-22).
Given that analog 22 maintains many structural features of the hit 1 as well as subsequent
molecules, it became incorporated into in vitro studies as an inactive control. Further
explorations with electron-donating methoxy groups in this region led to modest (24, 25) to
significant (23) losses in potency depending on the positioning. Lastly, in an attempt to boost
solubility, we combined the trifluoromethyl substituent with the pyridine in the most tolerant
position to generate analogs 26 and 27 (NCT-502). While NCT-502 (27) benefitted from a small
improvement in PHGDH potency, it did not benefit from an increase in kinetic aqueous
solubility (1.23 M at pH 7.4) as we expected. Despite its relatively poor aqueous solubility,
NCT-502 (27) proved to be a valuable tool molecule which was used to begin understanding the
in vitro biology of human PHGDH.
Table 4. Further SAR of the N-aryl piperazine of compound 15 and discovery of NCT-502 (27)
IC50(M)a
Cpd
R
% inhibition
92
6.3
15
8.7
101
18
4.3
37
98
73
19
20
Page 17 of 33

50
68
21
22
ND
33 (max)
24
109
23
14
16
11
3.7
101
99
24
25
26
100
96
27
(NCT-502)
aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay. ND = not determined; for compounds with % inhibition at 57 M
weaker than 40%, IC50 was ND.
With insufficient improvements in target potency and in vitro ADME properties through early
SAR and SPR studies, we considered more significant modifications to the piperazine core in
conjunction with changes to the group bridging to the trifluoromethyl phenyl ring. Along these
lines, we installed a one-carbon spacer (both methylene and carbonyl) from the piperazine core
to the arene segment. Additionally, we excised the piperazine nitrogen distal to the thiourea to a
pair of amino piperidines (both 3- and 4-substituted). Methods to access many of these
differentially substituted cores were reported previously.8, 19 The majority of the piperazine
analogs maintained much of their activity (15, 19, 30, 31) with significant drop-offs being
observed only with amides 28 and 29 (Table 5). The optimal 3-amino piperidine analog was that
of the p-trifluoromethyl aniline 33. The other changes to this core (32, 34-36) resulted in modest
decreases in activity. A similar trend was observed for the 4-amino piperidine core, with analogs
37 and 38 proving to be the best among the set, and analogs 39-41 showing losses in activity.
With regard to the three linkers, benzoyl led to a moderate drop in potency across all three cores.
Interestingly, the benzyl linkage proved optimal with the original piperazine core (analogs 30
and 31 [NCT-503]) as more significant drops in potency were observed with the piperidine ring
system (compounds 36 and 41). Lastly, the piperazine core was chosen for further SAR
evaluation as the solubility of top analog, 31 [NCT-503] (aqueous solubility = 25.3 M at pH
Page 18 of 33

7.4), was much greater than that of the top analogs from each of the amino piperidine sets
(analogs 33 and 37 displayed aqueous kinetic solubilities below the limits of quantitation).
Table 5. Core SAR of compound 15 and discovery of NCT-503 (31)
IC50(M)a
Cpd
R
% inhibition
92
m-CF3Ph
6.3
15
4.3
22
98
101
85
p-CF3Ph
19
28
29
30
m-CF3benzoyl
p-CF3benzoyl
m-CF3benzyl
19
5.3
102
31
p-CF3benzyl
2.5
96
(NCT-503)
m-CF3Ph
7.2
84
32
p-CF3Ph
5.7
10
85
91
88
89
33
34
35
36
m-CF3benzoyl
p-CF3benzoyl
p-CF3benzyl
8.7
12
m-CF3Ph
6.5
90
37
p-CF3Ph
7.7
11
10
15
97
94
90
93
38
39
40
41
m-CF3benzoyl
p-CF3benzoyl
p-CF3benzyl
Page 19 of 33

aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay.
To further probe the ethylene diamine feature of the core, a subset of ring-opened analogs were
evaluated. The increased flexibility resulted in a loss of activity with the dimethylated analog 44
maintaining moderate potency (Table 6). Conversely, rigid variations of the core, such as the
fused bis-pyrrolidine analog 45 and 1,4-diazepane 46, were also less active than NCT-503 (31).
Returning to the piperazine and the methylene linkage to the trifluoromethyl arene as above, we
Page 20 of 33

wanted to investigate whether substitution (47) or extension (48) would improve potency.
Unfortunately, both changes resulted in diminished activity. Furthermore, similar to much of our
previous SAR, piperazine substitution with both hydrophobic groups (49-52, 55) as well as
hydrophilic groups (53, 54) proved deleterious, with analogs 49 and 53 experiencing only small
decreases in PHGDH inhibition (Table 7).
Table 6. Piperazine SAR of compound 31 (NCT-503)
IC50(M)a
9.7
Cpd
42
R
% inhibition
p-CF3benzyl
p-CF3benzyl
p-CF3benzyl
41
66
84
21
43
7.3
44
p-CF3benzyl
p-CF3benzyl
16
7.3
8.0
23
93
82
90
87
45
46
47
48
aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay.
Table 7. Piperazine ring substition SAR of compound 31 (NCT-503)
Page 21 of 33

IC50(M)a
8.3
Cpd
49
% inhibition
99
94
86
12
50
9.9
51
ND
16 (max)
52
5.8
33
31
89
93
53
54
55
111
aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay. ND = not determined; for compounds with % inhibition at 57 M
weaker than 40%, IC50 was ND.
With the final phase of our SAR efforts focusing on a systematic exploration of the piperazine
aryl group, we hoped a thorough R group scan may provide a breakthrough in terms of target
potency. The general methodology we employed for this stage of the SAR campaign is
highlighted in the context of the synthesis of NCT-503 (31) (Scheme 1).1 Selective N-
benzylation of Boc-protected piperazine, protecting group removal, and subsequent one-pot
coupling to in situ-generated N-(4,6-dimethylpyridin-2-yl)-1H-imidazole-1-carbothioamide
proved direct and efficient. Thus, we scanned the positioning of many common aromatic
substituents about this ring (Me, OMe, F, Cl, CF3) without improvements in PHGDH activity
(Table 8). Testing of this large set of analogs focused on this aryl region of the chemotype
proved unable to improve potency.
Scheme 1. Methodology used to synthesize benzyl-substituted piperazines
Page 22 of 33

Table 8. Systematic SAR of benzyl piperazine portion of the pharmacophore
IC50(M)a
Cpd
R
% inhibition
phenyl
40
50
ND
59
9.2
20
17
ND
30
26
41
39
38
20
11
78
51
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
2-pyridyl
3-pyridyl
34 (max)
67
4-pyridyl
o-tolyl
85
m-tolyl
92
p-tolyl
93
o-anisoyl
21 (max)
94
m-anisoyl
p-anisoyl
87
o-fluorophenyl
m-fluorophenyl
p-fluorophenyl
o-chlorophenyl
m-chlorophenyl
56
88
86
106
90
Page 23 of 33

p-chlorophenyl
15
6.3
5.3
97
92
71
72
30
o-trifluoromethylphenyl
m-trifluoromethylphenyl
102
31
p-trifluoromethylphenyl
2.5
96
(NCT-503)
p-bromophenyl
17
11
92
96
73
p-trifluoromethoxyphenyl
74
aIC50 values were determined utilizing the diaphorase and resazurin-coupled
PHGDH assay. ND = not determined; for compounds with % inhibition at 57 M
weaker than 40%, IC50 was ND.
Discussion
The assay design and optimization described here outlines a novel coupled PHGDH assay with
robust performance and improved enzymatic turnover. This protocol was developed to avoid
many of the fluorescent artifacts and issues common to classic NADH-linked dehydrogenase
detection methods. This novel assay enabled a large-scale ultra high-throughput screen that
yielded validated small molecule PHGDH inhibitors, including one particular chemotype of
interest. The systematic structural exploration and optimization of this class of 2-pyridinyl-N-(4-
aryl)piperazine-1-carbothioamides with parallel SAR and SPR campaigns working in concert led
to the discovery of NCT-503 (31) as a selective probe for the detailed evaluation of human
PHGDH biology both in vitro and in vivo. As previously reported, NCT-502 (27) and NCT-503
(31) reduced the production of glucose-derived serine in cells and suppressed the growth of
PHGDH-dependent cancer cells, both in culture and in orthotopic xenograft tumors. The
metabolic effects of these molecules were found to be largely specific to PHGDH and
downstream pathways linked to serine flux. However, NCT-502 (27) inhibitor treatment was
found to reduce aspartate levels in cells, irrespective of PHGDH levels; decreased oxygen
consumption was observed in the presence of both active and inactive representatives of this
chemotype, suggesting the effect may be driven in part by electron transport chain inhibition.
This is suggestive of potential off-target effects by this scaffold, though more work needs to be
done to clearly delineate the implications of this activity. Importantly, this work demonstrated
that PHGDH inhibition by these probes reduced the incorporation of one-carbon units from
glucose-derived and exogenous serine into nucleotides. This observation suggested that
Page 24 of 33

glycolytic serine synthesis may coordinate the use of one-carbon units from serine in nucleotide
synthesis; thus, one-carbon unit wasting thus may contribute to the activity of PHGDH inhibitors
both in vitro and in vivo. Emerging from a structural class originally discovered and optimized
for the inhibition of bacterial phosphopantetheinyl transferase (PPTase), this scaffold was
selectively repurposed and optimized for PHGDH activity. Along these lines, a panel of small
molecules synthesized as part of PPTase SAR campaign was tested against both enzymes, and no
correlation in activity was observed between the two targets (Figure 5). In addition to an increase
in potency over both the hit molecule and that of NCT-502 (27), moderate improvements in both
solubility and microsomal stability were also observed for NCT-503 (31) (Table 9).
-4
-4.5
-5
-5.5
-6
-6
-5.5
-5
-4.5
-4
PHGDH Log(IC50)
Figure 5. Correlation plot showing activity (IC50s) of a panel of analogs against both PHGDH
and PPTase.
Table 9. Potencies and ADME properties of PHGDH modulators
PHGDH-Hit (1)
NCT-502 (27)
NCT-503 (31)
PHGDH-Inactive (22)
PHGDH IC50 (µM)
15.3
3.7 ± 1.0 (n=8)
2.5 ± 0.6 (n=4)
>57.0 (n=4)
Cytotoxicity EC50 (µM)
(MDA-MB-468)
-
15.2
8
-
Page 25 of 33

Pathway flux EC50 (µM)
(MDA-MB-468)
-
17.5
2.3
-
Kinetic Solubility (µM, in
buffer)
-
662.2 ± 39.8 (n=3)
1.2 ± 1.2 (n=8)
>30 (n=10)
61.5 ± 2.0 (n=3)
25.3 ± 2.6 (n=8)
>30 (n=8)
251.4 ± 24.9 (n=3)
>48 (n=2)
Aqueous Solubility (µM)
19
24
Rat Microsome Stability (t1/2,
min)
>30 (n=2)
These tool compounds complement an existing set of hPHGDH probes3 (Figure 1) discovered
through a variety of techniques including high-throughput screening6, fragment-based lead
generation5, structure-based drug design10, as well as a merger of both fragment screening and
pharmacophore convergence11. This class of inhibitors has expanded our understanding of the
role of PHGDH in tumor metabolism.
Experimental Procedures
Materials
3-Phosphoglycerate (P8877), NAD+ (N0632), glutamate (49621), DL-glyceraldehyde 3-
phosphate (G5251), glycerol-3-phosphate (G7886), TCEP (646547), C. kluyveri diaphorase
(D5540), and resazurin (R7017) were from Sigma.
Protein overexpression and purification
PHGDH, PSAT1 and PSPH were expressed and purified as previously described in Pacold et al,
Nature Chemical Biology 12, 452–458 (2016).
cDNAs to human PHGDH, PSAT1 and PSPH were PCR amplified from human liver cDNA
prepared from human liver mRNA using SuperScript III (Life Technologies) and cloned into
pET30-2 with an N-terminal 6xHis Tag. Proteins were expressed in Rosetta (DE3)pLysS E.
coli (EMD Millipore) grown to an OD of 0.6 and induced with 1 mM IPTG for 16 h at 16 °C.
Bacteria were lysed at 4 °C in a French press and purified by Ni2+ affinity chromatography on a 5
mL HiTrap chelating HP column (GE Healthcare) attached to an AktaPURE FPLC system (GE
Healthcare) using a gradient of 0–500 mM imidazole in 50 mM Na-Phosphate pH 8 and 300 mM
NaCl. Peak fraction purity was assessed by SDS gel electrophoresis. Pure fractions were
combined, concentrated in 15 mL UltraFree 30 concentrators (EMD Millipore) and loaded onto a
Page 26 of 33

HiLoad Superdex 200 prep grade 16/60 column equilibrated in 20 mM Tris pH 7.4, 100 mM
NaCl, and 1 mM TCEP. Peak fractions were concentrated to [protein] ≥ 5 mg/mL, flash frozen in
liquid nitrogen, and stored at −80 °C before use.
Enzyme assays
PHGDH assay buffer contained 50 mM TEA pH 8.0, 10 mM MgCl2, 0.05% bovine serum
albumin (BSA), and 0.01% Tween-20. PHGDH enzyme buffer consisted of assay buffer with 10
nM PHGDH and 0.15 mM NAD. PHGDH substrate buffer contained assay buffer with 10 µM
EDTA, 0.625 mM glutamate, 0.05 mM 3-phosphoglycerate, 0.1 mM resazurin, 500 nM PSAT1,
500 nM PSPH and 0.1 mg/mL diaphorase. The PHGDH primary assay was performed in 1,536-
well black solid-bottom plates. Substrate buffer was dispensed into each well (3 µL/well) using
a BioRAPTR Flying Reagent Dispenser (Beckman Coulter), and 23 nL of DMSO-solubilized
compounds were transferred into each well using a Kalypsys 1,536-well pintool equipped with
20 nL notched transfer pins. PHGDH enzyme buffer was then dispensed into each well (1
µL/well) and plates were spun in a plate centrifuge for 10 seconds to mix reagents begin the
reaction. Plates were stored at room temperature and resulting fluorescence was read at 0 min
and 20 min using a ViewLux uHTS Microplate Imager (PerkinElmer), using a 525/20 excitation
and 598/25 emission filter with a 5 sec exposure and excitation energy of 5000.
The uncoupled variation of the PHGDH assay was optimized to run in the absence of PSAT1 and
PSPH downstream enzymes.
PHGDH inhibition data was analyzed by calculating delta RFUs (increase in resorufin
fluorescence between T = 0 and T = 20 min) and normalizing to the delta RFU values of 0× and
1× PHGDH enzyme controls as 100% and 0% PHGDH inhibition, respectively. 32 wells of each
1536-well assay plate were dedicated to each of these 0× and 1× PHGDH controls. These
normalized dose-response values were plotted in GraphPad Prism and fit using a Sigmoidal
dose- response (variable slope) equation. IC50s for each replicate were then averaged to
determine average IC50s with SD.
General Synthetic Methods
All air- or moisture-sensitive reactions were performed under positive pressure of nitrogen with
oven-dried glassware. Anhydrous solvents such as dichloromethane, N,N-dimethylformamide
Page 27 of 33

(DMF), acetonitrile, methanol, and triethylamine were purchased from Sigma-Aldrich.
Preparative purification was performed on a Waters semi-preparative HPLC system. The column
used was a Phenomenex Luna C18 (5 micron, 30 x 75 mm) at a flow rate of 45 mL/min. The
mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A
gradient of 10% to 50% acetonitrile over 8 min was used during the purification. Fraction
collection was triggered by UV detection (220 nM). Analytical analysis was performed on an
Agilent LC/MS (Agilent Technologies, Santa Clara, CA). Purity analysis was determined using a
7 min gradient of 4% to 100% acetonitrile (containing 0.025% trifluoroacetic acid) in water
(containing 0.05% trifluoroacetic acid) with an 8 min run time at a flow rate of 1 mL/min. A
Phenomenex Luna C18 column (3 micron, 3 x 75 mm) was used at a temperature of 50 °C using
an Agilent Diode Array Detector. Mass determination was performed using an Agilent 6130
1
mass spectrometer with electrospray ionization in the positive mode. H NMR spectra were
recorded on Varian 400 MHz spectrometers. Chemical shifts are reported in ppm with non-
deuterated solvent (DMSO-h6 at 2.50 ppm) as internal standard for DMSO-d6 solutions. All of
the analogs tested in the biological assays have a purity greater than 95% based on LCMS
analysis. High-resolution mass spectrometry was recorded on Agilent 6210 Time-of-Flight
LC/MS system. Confirmation of molecular formulae was accomplished using electrospray
ionization in the positive mode with the Agilent Masshunter software (version B.02).
Experimental procedures
N-(4,6-dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide (22)
To a solution of di(1H-imidzazol-1-yl)methanethione (1.64 g, 9.19 mmol) in THF (24 mL) was
added 4,6-dimethylpyridine-2-amine (1.12 g, 9.19 mmol). The resulting reaction mixture was
heated with stirring at 40 °C for 35 min. During the reaction, the mixture was sonicated in order
to produce a homogeneous yellow slurry. To the resulting mixture was added 1-(pyridin-4-
yl)piperazine (1.50 g, 9.19 mmol). The resulting reaction mixture became red and was heated
with stirring at 50 °C for 2 h, then at 70 °C for 0.5 h. The reaction mixture was diluted with
water and dichloromethane. The layers were separated and the aqueous layer was re-extracted
with dichloromethane. The combined organic layers were dried with MgSO4 and concentrated in
vacuo. The resulting residue was taken up in DMSO and purified via reversed phase column
chromatography (0 to 50% acetonitrile/water 0.1% TFA). The pure fractions were combined and
Page 28 of 33

most of the organic portion removed in vacuo. To the resulting mixture was added
dichloromethane as well as saturated aqueous sodium bicarbonate solution, in order to free base
the product. The layers were separated and the aqueous layer was re-extracted with
dichloromethane three additional times. The combined organic layers were dried with MgSO4
and concentrated in vacuo to afford N-(4,6-dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-
1
carbothioamide (22, 1.33 g, 44%) as a light yellow solid. H NMR (400 MHz, DMSO-d6) δ 9.71
(br s, 1H), 8.18 – 8.12 (m, 2H), 7.20 (s, 1H), 6.81 – 6.70 (m, 3H), 4.05-3.95 (m, 4H), 3.47 – 3.39
13
(m, 4H), 2.33 (s, 3H), 2.21 (s, 3H); C NMR (101 MHz, DMSO-d6) δ 181.60, 155.19 (br),
154.29, 154.03, 150.25, 148.45 (br), 119.27, 115.87, 108.47, 47.93, 45.06, 23.47, 21.07; HRMS:
m/z (M+H)+ = 328.1597 (Calculated for C17H22N5S = 328.1590), Retention time: 1.953 min.
N-(4,6-dimethylpyridin-2-yl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-1-carbothioamide
(NCT-502, 27)
To a solution of di(1H-imidzazol-1-yl)methanethione (0.60 g, 3.4 mmol) in THF (15 mL) was
added 4,6-dimethylpyridine-2-amine (0.41 g, 3.4 mmol). The resulting reaction mixture was
heated with stirring at 40 °C for 30 min. During the reaction, the mixture was sonicated in order
to produce a homogeneous yellow slurry. To the resulting mixture was added 1-(5-
(trifluoromethyl)pyridin-2-yl)piperazine (0.78 g, 3.4 mmol). The resulting reaction mixture was
heated with stirring at 50 °C for 1 h. The reaction mixture was concentrated under a stream of
air. The resulting residue was taken up in DMSO and purified via reverse phase column
chromatography (acetonitrile/water 0.1% HCl). Combined fractions were partially concentrated
in vacuo, neutralized with saturated aqueous sodium bicarbonate solution, and filtered to remove
the solid. The solid was taken up in DMSO and repurified via reverse phase column
chromatography (5 to 100% acetonitrile/water 0.1% TFA). Combined fractions were partially
concentrated in vacuo (to remove organics) and filtered to provide the desired product, N-(4,6-
dimethylpyridin-2-yl)-4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-1-carbothioamide
1
trifluoroacetate salt (NCT-502, 27), 0.44 g, 26 %), as a solid. H NMR (400 MHz, DMSO-d6) δ
10.02 (br s, 1H), 8.42 (m, 1H), 7.82 (dd, J = 9.2, 2.6 Hz, 1H), 7.27 (s, 1H), 6.97 – 6.90 (m, 2H),
4.06 – 3.99 (m, 4H), 3.80 – 3.72 (m, 4H), 2.41 (s, 3H), 2.30 (s, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 181.34, 160.18, 158.81, 158.46, 145.68 (q, JC-F = 5.05 Hz), 135.06 (q, JC-F = 3.03
Hz), 126.65, 123.97, 120.6 (br), 116.93, 113.91 (q, JC-F = 32.32 Hz), 106.72, 48.21, 43.81, 22.05,
Page 29 of 33