Unravelling the allosteric targeting of phgdh at the act-binding domain with a photoactivatable diazirine probe and mass spectrometry experiments

Article abstract of DOI:10.3390/molecules26020477

The serine biosynthetic pathway is a key element contributing to tumor proliferation. In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this pathway, intensified and revealed to be a promising strategy to develop ne

Full text of DOI:10.3390/molecules26020477

molecules
Article
Unravelling the Allosteric Targeting of PHGDH at the
ACT-Binding Domain with a Photoactivatable Diazirine Probe
and Mass Spectrometry Experiments †
Quentin Spillier 1,2, Séverine Ravez 3, Simon Dochain 1, Didier Vertommen 4 , Léopold Thabault 1,2
,
Olivier Feron 2 and Raphaël Frédérick 1,
*
1
Medicinal Chemistry Research Group (CMFA), Louvain Drug Research Institute (LDRI),
Université Catholique de Louvain, B-1200 Brussels, Belgium; Quentin.Spillier@nyulangone.org (Q.S.);
simondochain@hotmail.com (S.D.); leopold.thabault@uclouvain.be (L.T.)
Pole of Pharmacology and Therapeutics (FATH), Institut de Recherche Expérimentale et Clinique (IREC),
Université Catholique de Louvain, B-1200 Brussels, Belgium; olivier.feron@uclouvain.be
UMR-S1172-JPArc-Centre de Recherche Jean-Pierre AUBERT Neurosciences et Cancer, Inserm, CHU Lille,
Université de Lille, F-59000 Lille, France; severine.ravez@univ-lille.fr
2
3
4
de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium;
didier.vertommen@uclouvain.be
*
Correspondence: raphael.frederick@uclouvain.be; Tel.: +32-2-764-73-41
†
This paper belongs to the Theme Issue in Honor of Prof. William A. Denny.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Abstract: The serine biosynthetic pathway is a key element contributing to tumor proliferation.
In recent years, targeting of phosphoglycerate dehydrogenase (PHGDH), the first enzyme of this
pathway, intensified and revealed to be a promising strategy to develop new anticancer drugs. Among
attractive PHGDH inhibitors are the α-ketothioamides. In previous work, we have demonstrated
their efficacy in the inhibition of PHGDH in vitro and in cellulo. However, the precise site of action
of this series, which would help the rational design of new inhibitors, remained undefined. In the
present study, the detailed mechanism-of-action of a representative α-ketothioamide inhibitor is
reported using several complementary experimental techniques. Strikingly, our work led to the
identification of an allosteric site on PHGDH that can be targeted for drug development. Using mass
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Spillier, Q.; Ravez, S.;
Dochain, S.; Vertommen, D.;
Thabault, L.; Feron, O.; Frédérick, R.
Unravelling the Allosteric Targeting
of PHGDH at the ACT-Binding
Domain with a Photoactivatable
Diazirine Probe and Mass
Spectrometry Experiments . Molecules
2021, 26, 477. https://doi.org/
10.3390/molecules26020477
spectrometry experiments and an original
identified the 523Q-533F sequence on the ACT regulatory domain of PHGDH as the binding site of
-ketothioamides. Mutagenesis experiments further documented the specificity of our compound at
α-ketothioamide diazirine-based photoaffinity probe, we
α
Academic Editor:
this allosteric site. Our results thus pave the way for the development of new anticancer drugs using
a completely novel mechanism-of-action.
Diego Muñoz-Torrero
Received: 31 December 2020
Accepted: 15 January 2021
Published: 18 January 2021
Keywords: PHGDH; diazirine; photoaffinity labeling
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Many recent findings highlighted the importance of serine metabolism in cancer [1–4].
Given that serine is a key metabolite to support cell proliferation, an increase in ser-
ine supply is required to sustain cancer progression. Serine can be taken up from the
extracellular environment or produced by the de novo serine synthesis pathway (SSP)
starting from the glycolytic metabolite 3-phosphoglycerate (3-PG). The SSP is composed
of three enzymes: phosphoglycerate dehydrogenase (PHGDH) that converts 3-PG into
3-phosphohydroxypyruvate, phosphoserine-amino transferase (PSAT-1) converting 3-
phosphohydroxypyruvate into phosphoserine, and phosphoserine phosphatase (PSPH)
eventually catalyzing the dephosphorylation of phosphoserine into serine.
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
In 2011, two independent publications highlighted the oncogenic role of PHGDH [
Since then, seminal publications confirmed the importance of PHGDH in cancer (triple
negative ER breast cancer, glioma, pancreatic cancer, etc.) [ ] and notably demonstrated
5,6].
7–9
Molecules 2021, 26, 477. https://doi.org/10.3390/molecules26020477
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 477
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that PHGDH extinction led to a significant reduction in tumor proliferation [10]. Given
the potential of PHGDH as an attractive anticancer drug target, research efforts were
devoted to identify potent PHGDH inhibitors (Figure 1) [10–17]. As depicted in Figure 1,
besides indole derivative developed by Astra Zeneca as NADH competitive inhibitors, all
reported molecules were shown to act as non-competitive inhibitors and are characterized
with PHGDH inhibitory potency in the micromolar range. Indeed, the high physiological
concentration of NADH (0.3 mM) hampers the design of competitive inhibitors. On
the other hand, the development of non-competitive, allosteric PHGDH inhibitors is a
promising approach, notably to overcome the problem of specificity against other NAD-
dependent enzymes.
Figure 1. Overview of some reported PHGDH inhibitors.
Until recently, only two different allosteric sites were identified for PHGDH, the
ASB (allosteric substrate binding) and the ACT (aspartate-kinase chorismate-mutase-tyrA)
domains. These two domains, located at the C-terminal part of the protein, have, up to
date, never been intentionally targeted to develop PHGDH inhibitors, and their role in
the control of PHGDH activity remains poorly understood. In 2016, Wang and coworkers
suggested two other allosteric sites of PHGDH that were confirmed by the use of probes
targeting these sites. The first, sharing at least five amino acids with the enzyme active
site (Gly 78, Val 79, Asp 80, Asn 81, and Val 82), is located at the interface of the enzyme
active site and NAD binding domain, whereas the second, smaller, was identified in the
substrate-binding cavity [13]. More recently, Zheng and coworkers suggested another
potential allosteric site located at the back-side of the active site and which could be the site
of action of the PHGDH inhibitor Ixocarpalactone A [17]. Finally, we have recently reported
an inhibition mechanism of PHGDH which involves disrupting its active oligomerization
state using disulfiram (DSF), a well-known anti-alcohol agent. DSF inhibits PHGDH
through oxidation of a specific cysteine (Cys116) located at the interface between two
PHGDH monomers [15].
These examples demonstrate the importance of detailing the mechanism-of-action of
newly developed PHGDH inhibitors to better understand the mechanisms involved in
PHGDH regulation and nurture the development of new inhibitors. We recently reported
a convergent pharmacophore strategy that led to the identification of the
(Figure 2) endowed with a PHGDH inhibitory potency in the 100
preliminary round of optimization around this hit led to the design of
α
-ketothioamide
M range [14]. A
exhibiting a
1
µ
2
five-fold improved IC50 value of 20.3 µM.

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Figure 2. α-ketothioamide inhibitors and preliminary SAR investigations.
In the present work, the site-of-action of compound
2 was investigated to identify
novel PHGDH allosteric site and possibly demonstrate that this site can be targeted to
design new anticancer therapies.
2. Results and Discussion
2.1. Biophysical Characterization of the Lead Compound
Previous studies in our laboratory highlighted compound
analogue as promising PHGDH inhibitors. Early investigations of the structure-activity rela-
tionships (SARs) revealed the importance of this para-substitution pattern (Figure 2, compare
the para-chlorinated derivative and the meta-chlorinated analogue , for instance) [14].
1 and the para-substituted
2
2
3
Moreover, recent studies also demonstrated that structural modifications of the linker
and/or the morpholino ring are poorly tolerated and only lead to improved inhibitory
potency when coupled with chlorinated para-substitution [18]. However, at this stage, the
precise binding mode of 2 to PHGDH remained mostly unclear. We previously reported
that this series act with a non-competitive mode of inhibition with respect to both substrates
and an apparently irreversible profile but without any evidence as to the location of this
potentially new allosteric site [14].
As a starting point of this study, we decided to detail the binding of
physical experiments. Microscale thermophoresis (MST) was thus used to appraise the
binding of to PHGDH. The experiments confirmed that 2 binds to PHGDH with a Kd
of 35.02 M, in the same range as the experimentally determined IC50 value. In contrast,
the meta-substituted analogue , used as a negative control in this experiment, showed no
appreciable binding (Figure 3).
2 using bio-
2
µ
3
Figure 3. Measurement of binding affinity between
PHGDH protein. Binding affinities (Kd) of and
performed in triplicates at each compound dilution, in brackets: 95% confidence intervals).
2
,
3
and PHGDH by MST. Dose-response curves of
2 and 3 on isolated
2
3 (Kd values were determined from two independent experiments
Having characterized the interaction of this inhibitor with the enzyme by MST, we
then turned our attention to the identification of the site-of-action. Because our attempt
to crystallize PHGDH with proved, in our hands, to be unsuccessful, a photo-reactive
crosslinking strategy was considered.
2

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2.2. Design and Synthesis of the Photoactivatable Probe 11
The reactive photoactivatable probe 11 was designed based on our initial SAR study
indicating that the para-position on the aryl moiety is preferred. Therefore, we envisaged
to incorporate the well-known photoactivatable trifluoromethyl diazirine moiety in this
particular para-position using the synthetic pathway depicted on Scheme 1 [19].
◦
Scheme 1. Synthetic pathway of diazirine 11. (
a
) ethylene glycol and PTSA.3H2O, toluene, 110 C, 24 h, quant. (
b
) (i) n-BuLi,
◦
◦
◦
THF,
) pTsCl, Et3N, DMAP, CH2Cl2, rt, 18 h, quant. (
H2O/acetone, rt, 18 h, 50%. (f) Br2, TBAB, CHCl3, rt, 18 h, 89%. (g) S8, morpholine, DMF, rt, 48 h, 82%.
−
78 C, 2 h (ii) ethyl trifluoroacetate, THF,
−
78 C, 3 h, 31%. (
c) NH2OH.HCl, NaOH, EtOH, 78 C, 18 h, 96%.
(
d
e) (i) NH3/MeOH, THF, rt, 18 h. (ii) I2, Et3N, MeOH, rt, 1 h. (iii) PTSA,
The chemical synthesis of the target molecule 11 started with the protection of the
commercially available ketone to its dioxolane (Scheme 1). Then, a two-step procedure
consisting of a lithium/halogen exchange followed by addition of ethyl trifluoroacetate
afforded the trifluoromethyl ketone . Condensation of with hydroxylamine and subse-
quent tosylation led to the tosyl-hydrazone intermediate . The diazirine was further
obtained by reacting with ammonia followed by oxidation and deprotection of the ketone.
Finally, the bis- -bromination of led to the dibromoketone 10 that was treated under the
4
5
6
6
8
9
8
α
9
Willgerodt–Kindler conditions to deliver the photoactivable probe 11 in good yields.
2.3. Evaluation of the Photoactivable Probe 11
Before investigating a possible adduct between the photoactivatable probe 11 and
PHGDH, its PHGDH inhibition potency was evaluated, and an IC50 value of 571
obtained (Figure 4). This lower PHGDH inhibitory potency compared to the parent
ketothioamide is relatively consistent with the previously identified SARs highlighting the
µM was
α-
2
limited possible pharmacomodulations in this position. Still, we hypothesized that although
less potent than 2, this tool compound could help us to reveal the binding mode in this series.
Figure 4. Dose–response curves of compounds
1, 2, and 11 on isolated PHGDH. IC50 values were determined from two
independent experiments performed in triplicates at each compound dilution, in brackets: 95% confidence interval.

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2.4. Epitope Mapping Analysis
The binding of the photoactivatable diazirine probe 11 with PHGDH was confirmed
by epitope mapping carried out by NMR saturation transfer difference (STD) experiments.
As depicted in Figure 5, both the photoactivatable diazirine probe 11 and the para-
chloro substituted compound 2 underwent saturation transfer from the protein via spin
diffusion through the nuclear Overhauser effect, corroborating their binding to PHGDH.
Furthermore, epitope mapping of both molecules demonstrated a similar pattern of inter-
action. Epitope mapping corresponds to the relative saturation transfer intensity between
the different protons of the molecule. Briefly, the ligands hydrogens in close contact with
the protein will undergo a higher saturation transfer than the protons less involved in
the interaction and hence display a more intense STD spectrum. Both molecules thus
displayed similar binding epitopes, with a high relative saturation transfer for the aro-
matic hydrogens (Figure 5, blue and red) and a lower relative saturation transfer for the
morpholine hydrogens (Figure 5, light blue and green) suggesting a similar interaction
pattern for 2 and 11 with PHGDH. This interesting pattern also explains, at least in part,
why modification/substitution of this aromatic ring has a considerable impact on PHGDH
inhibition, as previously reported [18].
Figure 5. Epitope mapping of 11 and
(bottom, 100 M) with PHGDH (10
2
. Superposition of 1H (blue) and STD (orange) spectra of
µM). The percentages correspond to the relative integration of the STD spectra compared
2 (top, 100 µM) and 11
µ
to the ones of the 1H spectra. All spectra are scaled in regard to the aromatic protons. A reduction of the signal intensity in
the STD spectrum compared to the 1H spectrum indicates a lower saturation transfer and hence a weaker interaction.
2.5. Photoactivation and Mass Spectrometry Analysis
Photoactivation experiments were performed with the diazirine 11 in the presence or
absence of 2 at various concentrations to analyze the potential competition for the same
binding site of these two compounds.
Briefly, PHGDH was incubated with or without
2 for one hour, and then the diazirine
derivative 11 (1 mM) was added for 30 min. The labeling with the photoactivatable diazirine
was performed by irradiation at 350 nm for 10 min. Finally, the enzyme-inhibitor complex

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underwent trypsin digestion, and the resulting peptides were analyzed by nanoUPLC/MS.
As a result, several peptides were identified, some of them being covalently modified
by the diazirine 11 (see Table S1 in Supporting Information). Interestingly, among the
detected peptides, only the 523QHVTEAFQFHF533 C-terminal peptide, that is part of the
ACT (aspartate kinase-chorismate mutase-TyrA) domain [20] of PHGDH (Figure 6A), could
be titrated out by addition with the competing compound
2. Indeed, only the percentage
of labeling of this particular peptide was reduced (from 43% to 6%) upon addition of
2,
thus suggesting that this particular peptide sequence is most probably part of an allosteric
binding site for the α-ketothioamide 2 (Table S2 in Supporting Information).
Figure 6. Result of the photoactivation experiment. (
with compound at different concentrations determined from the peptide spectrum matches (PSMs) of their precursors after
trypsinization and analysis by nanoUHPLC/MS. Compound was incubated with PHGDH in 50 mM Tris and 1 mM EDTA
at pH 8.5 during 1 h before adding 1 mM of 11. ( ) Schematic structure of full-length human PHGDH with substrate-binding
A) Percentage of the modified QHVTEAFQFHF peptide after incubation
2
2
B
domain in green, nucleotide-binding domain in blue, regulatory domains in red and diazirine modified peptide in purple.
Adapted from Ravez et al. [10].
To the best of our knowledge, this ACT domain in PHGDH has never been reported
before as a potential target binding site for PHGDH. Interestingly, in lower organisms like
E. Coli, this domain is a regulatory domain where L-serine binds to induce a negative feed-
back inhibition of the enzyme (Figure 6B). However, in human PHGDH (Type I enzyme),
this serine inhibition no longer occurs due to mutations of key binding residues [20]. The
allosteric inhibition by our α-ketothioamides, even if they act on the ACT domain, should
therefore be linked to a different and novel mechanism.
2.6. Protein Truncation Experiments
Finally, to further document our hypothesis that
2 binds to the allosteric site on
PHGDH delineated by the 523QHVTEAFQFHF533 C-terminal peptide, that is part of the
ACT domain, truncation experiments were conducted. To this end, a PHGDH truncated
form lacking the 523QHVTEAFQFHF533 C-terminal site (PHGDH-tr) was produced. Inter-
estingly, this mutant retained its enzyme activity in the same range to that of the native
PHGDH. However, the inhibitory potency of
abolished (Figure 7), hence demonstrating that the 523QHVTEAFQFHF533 C-terminal site
is indeed most probably the target of . As a comparison, disulfiram (DSF), a well-known
2 on the mutant was found to be completely
2
PHGDH inhibitor acting at an allosteric site distant from the site identified herein [15],
displayed the same PHGDH inhibitory potency both for the native and truncated enzymes.

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Figure 7. Dose–response curves of compounds 2 and DSF on isolated PHGDH (WT and tr). IC50 values were determined
from two independent experiments performed in triplicates at each compound dilution, in brackets: 95% confidence interval.
3. Materials and Methods
General Chemistry—All reagents were purchased from chemical suppliers and
used without purification. The characterizations of the products were performed as
reported previously [14].
PHGDH Assay—Enzymatic assay was carried out following a previously described
procedure [18].
PHGDH-tr Purification—pET28a human PHGDH-tr (Genecust) were transformed
into BL21 Escherichia coli, the protein was produced and tested following a reported
procedure [15]. Protein purity was assessed via SDS/PAGE and Coomassie staining.
MST assay—MST measurements were adapted from a previously reported proce-
dure [21]. Measurements were performed in 50 mM Tris buffer pH 8.5, 250 mM NaCl,
10 mM, MgCl2, and 100 µM DTT containing 0.05% P-20 in premium-treated capillaries
(NanoTemper Technologies, Munich, Germany). Experiments were performed in triplicates
using 40% LED power, medium MST power, LaserOn time was 20 s, Laser Off time 3 s.
Diazirine irradiation—Human PHGDH (5
µg) was incubated 30 min at room tem-
perature with compound 11 and compound at various concentrations in classical assay
2
buffer (50 mM Tris, pH 8.5, and 1 mM EDTA). The mixture was then irradiated on ice using
Stratalinker UV Crosslinker 1800 at 350 nm for 10 min.
Mass spectrometry experiments, Orbitrap Lumos—Mass spectrometry experiments
were carried out on an Orbitrap Lumos, following a previously reported procedure, with
some modifications [15]. Oxidation on Met; carbamidomethyl (+57.021 Da) on Cys and
diazirine (+315.068) on Cys, His, Phe, and Met were considered as variable modifications.
All modified peptide sequences were manually validated.
Epitope mapping by Nuclear Magnetic Resonance—All experiments were adapted
from a previously published procedure [21]. For 1D Saturation transfer difference (STD)
studies, samples were prepared in PBS with 5% d6-DMSO. The concentration of PHGDH
was 10 µM. Ligand binding was detected using a STD stddiffesgp.3 sequence with a 2 s
saturation time. For each experiment, 256 scans were collected both for on and off resonance
experiments (respectively at 0 and 40 ppm).
4. Conclusions
In this study, we aimed to detail the site of action of a series of α-ketothioamides
on PHGDH to fuel the development of new and potent anticancer drugs acting along a
potentially completely novel mechanism-of-action. First, we characterized the binding of
our lead compound 2 using microscale thermophoresis experiments. Then, a crosslinking
strategy using an original photoactivatable diazirine probe was used to decipher the
binding of 2 on PHGDH. Interestingly, this led to the identification of a previously unknown
allosteric site on PHGDH delineated by the 523QHVTEAFQFHF533 C-terminal peptide
sequence, that is part of the PHGDH ACT domain, as the target binding site. We further
confirmed our hypothesis using a complementary protein truncation experiment.

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Although to our knowledge, the structure of the allosteric site identified here is not
known, our data pave the way to the development of novel anticancer agents acting along
a particularly original mechanism-of-action.
Supplementary Materials: The following are available online: Chemical synthesis and characteri-
zation for 1–11. Identified peptide residues of PHGDH before and after titration. NMR and HPLC
spectrum for 2 and 11.
Author Contributions: Q.S., S.R., R.F. and O.F. designed the experiments, analyzed the data, wrote
and edited the manuscript. Q.S. performed all experiments except the synthesis of 11 (done by S.D.),
MS analysis (done by D.V.) and the Epitope mapping (done by L.T.). Q.S. and L.T. prepared the
figures. R.F. supervised all aspects of the project. The manuscript was written through contributions
of all authors. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by a J. Maisin Foundation grant, the Fondatioun Kriibskrank
Kanner (Luxembourg), a Credit de Recherche (CDR) grant from the F.R.S.-FNRS, and an Action de
Recherche Concertee (ARC 14/19-058) grant from the Federation Wallonie-Bruxelles. Q.S. is Televie
Research Fellows.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We are grateful to Gaëtan Herinckx and Vincent Stroobant for their contribution
to this project and to the Nuclear and Electron Spin Technologies platform within the Louvain Drug
Research Institute (LDRI-UCLouvain) for access to NMR facilities.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 1, 2, 3, and 11 are available from the authors.
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