Towards the development of activity-based probes for detection of lysine-specific demethylase-1 activity

Article abstract of DOI:10.1016/j.bmc.2016.11.043

The implications of lysine-specific demethylase-1 (LSD1) in tumorigenesis have urged scientists to develop diagnostic tools in order to explore the function of this enzyme. In this work, we present our efforts on the development of tranylcypromine (TCP)-based functionalized probes for activity-based protein profiling (ABPP) of LSD1 activity. Biotinylated forms of selected compounds enabled dose-dependent enzyme labeling of recombinant LSD1. However, treatment with LSD1 inhibitors did not clearly reduce the LSD1 labeling efficiency thus indicating that labeling using these probes is not activity dependent. This calls for alternative strategies to develop probes for ABPP of the enzyme LSD1.

Full text of DOI:10.1016/j.bmc.2016.11.043

Accepted Manuscript
Towards the development of activity-based probes for detection of Lysine-Spe-
cific Demethylase-1 activity
Maria E. Ourailidou, Alessia Lenoci, Clemens Zwergel, Dante Rotili, Antonello
Mai, Frank J. Dekker
PII:
S0968-0896(16)31264-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.043
BMC 13410
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 August 2016
21 November 2016
23 November 2016
Please cite this article as: Ourailidou, M.E., Lenoci, A., Zwergel, C., Rotili, D., Mai, A., Dekker, F.J., Towards the
development of activity-based probes for detection of Lysine-Specific Demethylase-1 activity, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.043
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Bioorganic & Medicinal Chemistry
Towards the development of activity-based probes for detection of Lysine-Specific
Demethylase-1 activity
Maria E. Ourailidoua , Alessia Lenocib , Clemens Zwergelb, Dante Rotilib , Antonello Maib,c, and Frank J.
Dekkera, 
aDepartment of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1,
Groningen 9713 AV, The Netherlands
bDepartment of Drug Chemistry and Technologies, ‘Sapienza’ University, P.le A. Moro 5, 00185 Rome, Italy
cPasteur Institute, Cenci Bolognetti Foundation, ‘Sapienza’ University, P.le A. Moro 5, 00185 Rome, Italy
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The implications of Lysine-Specific Demethylase-1 (LSD1) in tumorigenesis have urged
scientists to develop diagnostic tools in order to explore the function of this enzyme. In this
work, we present our efforts on the development of tranylcypromine (TCP)-based functionalized
probes for activity-based protein profiling (ABPP) of LSD1 activity. Biotinylated forms of
selected compounds enabled dose-dependent enzyme labeling of recombinant LSD1. However,
treatment with LSD1 inhibitors did not clearly reduce the LSD1 labeling efficiency thus
indicating that labelling using these probes in not activity dependent. This calls for alternative
strategies to develop probes for ABPP of the enzyme LSD1.
Available online
Keywords:
Lysine demethylation
Tumorigenesis
Tranylcypromine
Irreversible inhibition
Enzyme labeling
20016 Elsevier Ltd. All rights reserved.
1. Introduction
substrates and interactions with other proteins, LSD1 is known to
mediate transcriptional activation or repression.9,10 Accumulating
Among post-translational modifications, histone lysine
methylation has gained increasing attention as a major regulator
of the transcriptional potential of eukaryotic cells. Lysine
residues in histone tails (mainly of histones H3 and H4) can be
mono-, di- or trimethylated on their ε-amino groups by lysine
methyltransferases (KMTs) resulting in different functional
outputs.1,2 Lysine demethylases (KDMs) act as erasers of these
marks and, depending on the enzymatic mechanism, they are
categorized to lysine-specific demethylases (LSDs) and
JumonjiC (JmjC) demethylases.3 The first class are FAD (flavin
adenine dinucleotide)-dependent amine oxidases that demethylate
mono- or dimethylated lysine residues producing formaldehyde
and hydrogen peroxide, whereas the second class utilizes Fe(II)
and α-ketoglutarate as cofactors and can also act on trimethylated
lysine side chains via its dioxygenase function.
data suggest that any imbalance of the dynamic regulation of
lysine methylation due to aberrant expression of LSD1 can cause
dramatic alterations in gene transcription and, consequently, in
the development and progression of various cancer types.11-14
Nevertheless, several studies demonstrate that, in coordination
with other proteins, LSD1 affects the growth of breast cancer
cells negatively,15-17 while promoting effects have been described
for viral infections.18,19
Due to its significant role in pathogenesis, LSD1 has been an
emerging pharmacological target and thus, the development of
potent inhibitors has attracted increasing research interest. Up to
date, a wide variety of compounds has been reported to inactivate
LSD1 in a reversible20-24 or irreversible way,25-27 which have been
evaluated mainly for their antiproliferative effects. The majority
of them was inspired by several anti-MAO (monoamine oxidase)
agents found to inhibit LSD1 with tranylcypromine (trans-2-
phenylcyclopropyl-1-amine, TCP) taking center stage. TCP is a
non-selective irreversible MAO A/B inhibitor with a reported
potency over LSD1 ranging from 20-400 µM.25,28 Structural and
kinetic studies revealed a suicide-mechanism of time-dependent
Two mammalian flavin-containing demethylases have been
identified: LSD1 (also known as KDM1A, AOF2, BHC110 or
KIAA0601) and LSD2 (KDM1B or AOF1).4,5 The best studied is
LSD1 which is now well documented to represent an important
player in developmental processes, embryogenesis and
differentiation of numerous cellular types.6-8 According to its
———
 Corresponding author. Tel.: +39 338 852 46 07; fax: +39 06 49693268; e-mail: antonello.mai@uniroma1.it
 Corresponding author. Tel.: +31 50 3638030; fax: +31 50 3637953; e-mail: f.j.dekker@rug.nl

inhibition via opening of the cyclopropyl ring and formation of a
stable covalent adduct with the reduced form of the cofactor
FAD.25,28 Although different models have been proposed
ensure for covalent attachment to the cofactor FAD. However,
unlike MAO and other flavoenzymes, the interactions between
FAD and LSD1 are not covalent and thus we hypothesized that
Figure 1. Irreversible enzyme binding through covalent linkage. (A) Proposed mechanism of LSD1 inactivation by racemic TCP.30 (B)
Photocrosslinking with benzophenone-type activity-based probes.
regarding the structure of this adduct,29 further structural analyses
and crystallographic data from LSD1-TCP complex indicated the
participation of the N(5) atom of the flavin ring (Fig. 1A).30,31
TCP has been employed by numerous research groups as the
starting point for the development of more potent and selective
derivatives with promising antitumor effects.29,32-38
detection via the ABPP method would not be possible. Therefore,
we firstly designed probe 8 where a benzophenone group was
introduced aiming at photocrosslinking and covalent binding to
the protein (Fig. 1B). Benzophenones are known to be activated
upon ultraviolet irradiation at long wavelengths to generate a
diradical that reacts irreversibly with neighboring C-H bonds, in
particular those of methionine residues. They are generally
characterized by fast activation and limited cross-reactivity.45-48
In addition, a polar linker was used to connect TCP with the
photoactivatable part, whereas a protruding propargyl group was
inserted to serve for subsequent linkage to a detection label via
the ‘click reaction’ (Scheme 1).
Despite the tremendous progress on LSD1 inhibition, its
controversial roles in gene expression and oncogenesis call for
the discovery of novel diagnostic tools to gain a better insight on
the biological function of this enzyme.39 Activity-based protein
profiling (ABPP) has been proven to be a valuable approach to
study intracellular enzyme activity.40-42 In this work, we report
the design, synthesis and biological evaluation of activity-based
functionalized probes for detection of human recombinant and
endogenous LSD1.
Alternatively to probe 8, we developed three non-
benzophenone-bearing compounds as controls to test the need for
photocrosslinking. Recently Valente et al. developed derivatives
of TCP, with a benzoylamino group at the para, meta or ortho
position in respect to the phenyl ring.49 They observed that the
para-substituted compounds exhibited the greatest potency at
low nanomolar scale regarding MAO-A and LSD1 inhibition.
Inspired by this, we employed a para-benzoylamino TCP
analogue (Scheme. 2, compound 12) functionalized at position 4
(in respect to the benzoyl) with a terminal alkene and two
alkoylamino derivatives (compounds 13 and 14) functionalized
with terminal olefinic and a alkyl chains respectively. The
alkyne-labeled compounds allow for detection via the ‘click’
reaction while the olefinic ones via the oxidative Heck reaction,
recently introduced by our group as a successful bioorthogonal
strategy for in vitro protein labeling in aqueous environment.50,51
2. Results and discussion
2.1. Molecular design
Two different approaches were followed in the molecular
design of the probes. Activity-based probes are typically
designed in a way to mimic the covalent binding of the substrate
to the protein, modifying the latter in an irreversible manner.
Protein visualization is then achieved directly after labeling, in
case the probes contain a detection handle (i.e. fluorophore), or
after bioorthogonal coupling to affinity tags. For instance,
Breinbauer et al. developed functionalized probes based on the
irreversible MAO inhibitors pargyline and deprenyl for the
detection of MAO-A and B in tissues and live cells, while, later
on, selective imaging and profiling of MAO-B in a variety of
biological samples was achieved by Yao et al. employing a dual-
purpose functionalized activity-based probe.43,44 For our
purposes, all the developed compounds included a TCP moiety to

2.2 Chemical synthesis
All compounds were synthesized from tert-butyl (trans-2-(4-aminophenyl)cyclopropyl)carbamate (6) after coupling to the
corresponding carboxylic acids followed by acidic deprotection of the Boc group (Scheme 1). Regarding the synthesis of the
photocrosslinking and linker part of probe 8, compound 3 was generated after coupling of D-propargylglycine to 4-benzoylbenzoic
acid using HOBt/EDCI/triethylamine (TEA) in good yield (64%) (Scheme 1A). For the introduction of the TCP functional group,
compound
6
was first synthesized according to previously reported methods,30 including p- nitration of trans-2-
phenylcyclopropane-1-carboxylic acid (32%), Curtius rearrangement and Boc protection of the afforded cyclopropylamine (27%)
and, finally, reduction of the nitro group to the corresporesponding aniline (27%) (see Experimental section). Afterwards, Boc-
protected forms of probes 8, 12 and 14 were provided using EDC as a coupling reagent, while in the case of probe 13, the coupling
was performed with the use of pent-4-enoyl chloride and TEA (Scheme 1B).
2.3 Inhibition studies
All synthesized compounds were screened for inhibition against human recombinant LSD1. The principle of the in vitro assay
was based on the oxidative demethylation of the monomethylated histone peptide H3K4N(CH3) via a FAD/FADH2 mediated
reduction of O2 to H2O2. The remaining activity of LSD1 was monitored via detection of the amount of H2O2 formed. This is done
by Horseradish Peroxidase (HRP) which reduces H2O2 to H2O using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP, Amplex Red, for
Figure 3. IC50 values of developed LSD1 probes and their respective
standard deviations.
synthesis see Experimental section) as an electron donor. The resulting product (resorufin) is highly fluorescent at 590 nm (Fig. S1).
The inhibition assay was performed as described previously (see Experimental section).49 The compounds were pre-incubated at

different concentrations with LSD1 for 15 min at room temperature in presence of HRP-Amplex Red. The substrate was then added
and the fluorescence was measured for 30 min. The IC50 values were found to be in the high nanomolar range apart from compound
12 which exhibited a potency of 0.21 µM (Fig. 3).
We then moved to the determination of the kinetic
parameters Ki (equilibrium binding constant) and ki (inactivation rate
constant) that characterize the irreversible type of inhibition through Kitz-Wilson analysis (see Experimental section). At start,
irreversible inhibitors form a non-covalent complex with the enzyme (represented by Ki) which then reacts to generate the covalent
‘dead-end complex’ (represented by ki). For our purposes, the enzyme was incubated with different concentrations of each
compound for different incubation times in presence of HRP-Amplex Red. Substrate was added and the enzyme activity was
monitored as described above. In all cases, time-dependent inhibition was observed with Ki values ranging between 3-5 µM and the
ki values between 0.3-0.4 min-1 indicating the rapid binding of the TCP derivatives to the FAD cofactor (reported ki for TCP 0.67
min-1).52
2.4 Labeling studies
2.4.1
Human recombinant LSD1
Having all the information required on the appropriate range of inhibitor concentration and incubation time necessary for
binding, we set out to utilize them for labeling of human recombinant and cellular LSD1. Initial experiments included treatment of
HeLa cell lysates with various concentrations of each compound and subsequent coupling to azide-PEG3-biotin via the ‘click’
reaction (compound 14)51 or 3-(biotinylamino)phenylboronic acid via the oxidative Heck reaction (compounds 12 and 13).51
Unfortunately, after imaging using the enhanced chemiluminescence assay, only endogenously biotinylated proteins were
Figure 4. Detection of human recombinant LSD1. (A) Luminescence imaging after incubation with various concentrations of pre-clicked
biotinylated probes 17 and 18. (B) Control reactions. ΔT; heat-denaturation control, PC; positive control.
visualized, while the presence of LSD1 was confirmed with an anti-LSD1 antibody (data not shown). As supported from our
previous findings, where incomplete conversion was observed for both bioorthogonal reactions at protein concentrations lower than
5 µM,51 we speculated that no labeling was due to the limiting performance of the reactions at the low protein concentrations in the
case of cell lysates. Another reason may be that the reactive group was not properly positioned to react with the complementary
coupling reagents.
Therefore we continued our labeling efforts by employing a single-step process, assuring for the biotinylation step:
benzophenone probe 8 and probe 14 were firstly subjected to ‘click’ reactions and coupling to azide-PEG3-biotin (see Experimental
section) to generate the biotinylated compounds 17 and 18 accordingly. Human recombinant LSD1 was incubated with 0-40 µM of
each compound for 10 min. In case of probe 17, the incubation was followed by 5 min UV irradiation at 365 nm. The enzyme was
then visualized using anti-LSD1 antibody, proteins were stripped and subsequently detected via HRP-streptavidin. Surprisingly, a
concentration-dependent luminescent signal was observed not only for probe 17 (Fig. 4A) but also for the non-benzophenone-
carrying compound 18 (Fig. S3).. This finding is remarkable since previous studies where, in an attempt to prove the non-covalent
binding of FAD to the enzyme, TCP-inactivated protein was denatured in presence of 0.3% SDS resulting in release of FAD and
separation from the enzyme by HPLC.28 Our observation is difficult to explain at this point, nevertheless it indicates that
benzophenone cross-linking is not key to labelling of LSD1 using the TCP-based probe applied in this study. We therefore decided
to continue our studies using both probes 17 and 18 in order to explore which inhibitor-type is best for activity-based labeling.
Control reactions were performed to ensure for activity-based labeling. For the negative control experiment, the enzyme was
subjected to ‘click’ reaction conditions while, regarding the heat-denaturation control (ΔT), boiling for 15 min before labeling
served for complete unfolding, protein aggregation and loss of activity as reported before.28,53 Compound 17 was added to the heat-
denaturated and positive control samples at a final concentration of 40 µM and immediately all the samples were irradiated shortly
(5 min). Furthermore, in order to confirm that the TCP-moiety is crucial for labelling, we did a control experiment in which the the
TCP moiety was excluded by employing compound 2 that consists of a benzophenone and propargyl group but not the TCP

(Scheme 1). Analogously to the rest of the controls, LSD1 was irradiated for 5 min in presence of 40 µM of pre-clicked compound 2
and detected in all cases as described above. Pleasingly, labeling occurred only in the positive control sample demonstrating that
TCP is essential for enzyme labeling (Fig. 4B, Fig. S4). The same experiments were repeated for compound 18 (final concentration
10 µM) and pre-clicked compound 2 was used at a final concentration of 10 µM, where, again, luminescence was observed only in
the case of the positive control sample.
Figure 5. Detection of human recombinant LSD1 after treatment
with known inhibitors. The enzyme was pre-incubated with
compound 12 and TCP and subsequently treated with pre-clicked
probes 17 (A) and 18 (B). PC; positive control.
Next, in order to further certify an activity-based labeling, we investigated the protein tagging after treatment with known LSD1
inhibitors (Fig. 5, Fig. S5). Compound 12 (1 mM) and TCP (1.5 mM) were pre-incubated with a low concentration of LSD1 for 30
min at room temperature. Compound 17 was added at a final concentration of 40 µM and the samples were immediately irradiated.
The irradiation time was decreased from 5 to 2 min to induce the effect of the inhibitors. After detection via the usual procedure, no
reduction in protein labeling was visible, compared to the positive control (Fig. 5A). This can be justified for TCP, known for its
weak potency over LSD1 as mentioned above, but not for compound 12 whose equilibrium binding constant Ki was found to be 2.7
µM. The same experiment was repeated using probe 18 and only the potent compound 12 and, again, no clear difference was
observed between the inhibitor-treated samples and the positive control (Fig. 5B). These results point to a lack of enzyme activity-
dependence of the LSD1 labeling. However, as indicated by the control experiments, this can be attributed only to the TCP moiety
and not the triazole-PEG3-biotin or the photocrosslinking one
Figure 6. Labeling experiments on HeLa nuclear extracts. (A) Detection of endogenous proteins after treatment with various concentrations of
pre-clicked probes 17 and 18. (B) Control reactions and (C) treatment with LSD1 inhibitors. Proteins were detected by using HRP-streptavidin-
coupled enhanced chemiluminescence assay.

2.4.2. HeLa nuclear extracts
Afterwards, we moved on to test the labeling efficiency of probes 17 and 18 on HeLa nuclear extracts, known as a source of
endogenous LSD1.54,55 Various concentrations of each probe were incubated with protein extracts for 10 min followed by 5 min
irradiation (for 17) or for 15 min at room temperature (for 18). Subsequent detection revealed a number of biotinylated proteins,
potentially FAD-dependent (Fig. 6A, Fig. S6, S7). However, no visible labeling of LSD1 was observed, while its presence was
confirmed via an anti-LSD1 antibody. This may be due to low LSD1 activity after nuclear extraction or to probe utilization by other
enzymes, presumably amine oxidases. Additionally, it is possible that the probe does not bind to nuclear LSD1 as the enzyme exists
in complex with other cofactors.54,56 Similar control experiments took place on nuclear extracts as described for the purified
enzyme. Again, biotinylated proteins were visualized only in the case of the positive control sample, while heat-denaturated proteins
hardly reacted and treatment with the non-TCP-bearing pre-clicked compound 2 resulted in no tagging (Fig. 6B, Fig. S8). This
indicates that labeling occurs only in non-denatured enzymes and is due to the presence of TCP moiety.
Finally, HeLa nuclear extracts were pre-treated with 1 mM of compound 12 or 1.5 mM of TCP and then labeled with the
corresponding probe for 2 min. Nevertheless, no change in the level of biotinylation of endogenous proteins was shown upon
incubation with the inhibitors (Fig. 6C). These findings are in line with the results obtained from the purified LSD1 and indicate a
lack of activity-dependence in the observed labelling.
3. Conclusions
In conclusion, we have developed a series of alkene- and alkyne-labeled TCP-based probes for detection of LSD1 activity via
activity-based protein profiling. Considering the reversible interactions between the enzyme and the cofactor FAD, we also
designed a benzophenone-containing alkynic compound for covalent attachment to LSD1 after photocrosslinking with UV
irradiation. Alkyne-bearing probes 8 and 14 were subjected to ‘click’ reactions for linkage to a biotin molecule and incubated with
human recombinant LSD1. In both cases, a luminescence signal was observed while heat-inactivation or treatment with a non-TCP-
containing probe resulted in no labeling. This demonstrates that tagging occurs only to non-denatured LSD1 and that the
tranylcypromine group is crucial for enzyme inhibition. Unfortunately, we found no apparent reduction in labeling when LSD1
inhibitors were applied, a fact that implies that small molecule inhibition of the enzyme activity does not corroborate to the activity-
based labeling. Further studies on HeLa nuclear extracts indicated that several proteins were effectively labeled by the probes,
without a pronounced labeling of LSD1, while inhibition experiments, again, did not reveal a clear reduction in protein labeling.
According to our control experiments, the developed molecules can be used as activity-based probes based on non-denatured
enzymes but are disfavored in small molecule inhibition studies. Our data together demonstrate that photocrosslinking is not needed
for enzyme labeling. This work sets the stage for further elucidation of the mechanism of inhibition of TCP derivatives and should
be taken into account when designing novel analogues aiming at detecting LSD1 activity.
4. Experimental section
4.1. Chemistry
4.1.1. General
Chemicals were obtained from commercial suppliers (Sigma Aldrich, Acros Organics, Axon Medchem) and used without further
purification, unless stated otherwise. Aluminum sheets of Silica Gel 60 F254 were used for Thin layer chromatography (TLC).
Spots were visualized under ultraviolet light or stained with KMnO4 solution. MP Ecochrom Silica Gel 32-63 60 Å was used for
flash column chromatography. NMR spectra were recorded on a Bruker Avance 500 spectrometer (1H NMR; 500 MHz), 13C NMR;
125 MHz)). Chemical shift values are reported in ppm (δ) relative to tetramethylsilane (TMS). Coupling constants (J) are reported
in Hz with the following splitting abbreviations: s = singlet, d = doublet, t = triplet, q = quartet and m = multiplet. Abbreviations are
defined as follows: dichloromethane (DCM), methanol (MeOH), thionyl chloride (SOCl2), dimethylformamide (DMF), N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI), 1-hydroxybenzotriazole hydrate (HOBt), trimethylamine (TEA), ethyl acetate
(EtOAc), diphenylphosphoryl azide (DPPA) and magnesium sulphate (MgSO4).
4.1.2 (R)-methyl 2-aminopent-4-ynoate (1)
To an ice-cooled solution of D-propargylglycine (0.30 g, 2.7 mmol, 1.0 eq) in MeOH (5.0 mL), thionyl chloride (0.40 mL, 40
mmol, 15 eq) was added dropwise. The resulting mixture was heated at 60 °C for 4h. After completion of the reaction, the solvent
was evaporated and the crude mixture was washed with DCM three times to obtain the pure compound 1 as a light brown oil (0.30
g, 89%). 1H NMR (500 MHz, CDCl3): δ = 8.73 (bs, 2H), 4.47 (s, 1H), 3.86 (s, 3H), 3.73 (m, 1H), 3.20-3.15 (m, 1H), 3.06-3.01 (m,
1H) ppm. 13C NMR (125 MHz, CDCl3): δ = 168.5, 76.4, 74.4, 53.7, 52.0, 20.6 ppm.
4.1.3 (R)-ethyl 2-(4-benzoylbenzamido)pent-4-ynoate (2)
To a solution of 4-benzoylbenzoic acid (0.30 g, 1.3 mmol, 1.0 eq) in DMF (3.5 mL) were added EDCI (0.33 g, 1.7 mmol, 1.3
eq), HOBt (0.27 g, 1.7 mmol, 1.3 eq) and TEA (0.65 mL, 4.6 mmol, 3.5 eq). After stirring for 30 min at room temperature, 1 (0.17
g, 1.3 mmol, 1.0 eq) was added and the mixture was stirred at room temperature for additional 17h. Then, the reaction was
quenched with NaHCO3 (20 mL) and extracted with EtOAc (3 x 20mL). The organic layer was washed with brine, dried over

MgSO4 and concentrated. The crude mixture was purified by flash chromatography on silica gel (EtOAc:petroleum ether 1:2)
affording compound 2 as a white solid (0.29 g, 64%). Rf = 0.45 (EtOAc:petroleum ether 1:1). 1H NMR (500 MHz, CDCl3): δ = 7.93
(d, J = 8.2 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 7.8 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.52-7.49 (m, 2H), 7.05 (d, J = 7.3
Hz, 1H), 4.99-4.95 (m, 1H), 3.85 (s, 3H), 2.99-2.88 (m, 2H), 2.09-2.08 (m, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ = 196.0,
170.9, 166.3, 140.7, 137.1, 137.0, 133.1, 130.3 (x2), 130.2 (x2), 128.6 (x2), 127.3 (x2), 78.4, 72.1, 53.2, 51.2, 22.7 ppm.
4.1.4 2-(4-Benzoylbenzamido)pent-4-ynoic acid (3)
Compound 2 (0.29 g, 0.89 mmol, 1.0 eq) was dissolved in THF:MeOH (2.0 mL:1.0 mL) and a solution of LiOH (31 mg, 1.3
mmol, 1.5 eq) in H2O (1.0 mL) was added at 0 °C. After 3h stirring at room temperature, LiOH (31 mg, 1.3 mmol, 1.5 eq) was
added. After further stirring for 5h, the mixture was acidified to pH 1.0 with a solution of HCl 1N and extracted with EtOAc (3 x 10
1
mL). The organic phase was then dried and evaporated to afford compound 3 as a white solid (0.27 g, 97%). H NMR (500 MHz,
CD3OD): δ = 7.99 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 7.4 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.56-7.53 (m,
2H), 4.78-4.76 (m, 1H), 3.31-3.30 (m, 1H), 2.94-2.81 (m, 2H), 2.39-2.38 (m, 1H) ppm. 13H NMR (125 MHz, CD3OD): δ = 193.8,
175.8, 174.2, 140.5, 139.3, 136.8, 134.2, 131.1 (x2), 131.0 (x2), 129.7 (x2), 128.6 (x2), 80.4, 72.1, 53.4, 22.2 ppm.
4.1.5 trans-2-(4-Nitrophenyl)cyclopropane-1-carboxylic acid (4)
In a 50 mL round bottom flask equipped with a stirring bar, trans-2-phenylcyclopropane-1-carboxylic acid (2.5 g, 15 mmol, 1.0
eq) was added. A solution of HNO3 (70% in H2O) (15 mL, 0.23 mol, 15 eq) was added dropwise at 0 °C. After 3h stirring at room
temperature, additional HNO3 (2.0 mL) was added and, after 3h, the mixture was filtrated and washed over the filter with H2O. The
crude mixture contained para:ortho nitro substituted product in a ratio 4:1. It was triturated in toluene and then filtrated. The solid
over the filter was washed with petroleum ether to afford compound 4 as a white solid (1.0 g, 32%). 1H NMR (500 MHz, CD3OD):
δ = 8.15 (d, J = 7.6 Hz, 2H), 7.37 (d, J = 7.6 Hz, 2H), 2.58-2.57 (m, 1H), 2.00-1.98 (m, 1H), 1.66-1.62 (m, 1H), 1.49-1.45 (m, 1H)
ppm. 13H NMR (125 MHz, CD3OD): δ = 176.1, 150.0, 147.9, 128 (x2), 124.7 (x2), 26.6, 26.1, 18.3 ppm.
4.1.6 tert-Butyl (trans-2-(4-nitrophenyl)cyclopropyl) carbamate (5)
To a suspension of compound 4 (0.50 g, 2.4 mmol, 1.0 eq) in dry toluene, TEA (0.40 mL, 2.9 mmol, 1.2 eq) and DPPA (0.60
mL, 2.8 mmol, 1.2 eq) were added dropwise at 0 °C. Then tert-butanol (3.5 mL, 36 mmol, 15 eq) was added at room temperature
and the mixture was heated at 105 °C. After 16h, di-tert-butyl-dicarbonate (Boc2O, 0.53 g, 2.4 mmol, 1.0 eq) was added at room
temperature and the mixture was stirred for another 2h at 105 °C. The reaction was quenched with H2O (5.0 mL) and extracted with
EtOAc (6 x 20 mL). The organic layers were combined, dried over MgSO4 and evaporated under reduced pressure. The crude
mixture was purified by flash chromatography on silica gel (EtOAc:petroleum ether 1:6) affording compound 5 as a yellowish solid
1
(0.18 g, 27%). Rf = 0.28 (EtOAc:petroleum ether 1:2). H NMR (500 MHz, CDCl3): δ = 8.12 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4
Hz, 2H), 4.92 (bs, 1H), 2.78 (m, 1H), 2.13 (m, 1H), 1.44 (s, 9H), 1.32-1.24 (m, 2H) ppm. 13C NMR (125 MHz, CDCl3): δ = 149.1,
146.4, 127.1, 123.8 (x4), 71.7, 33.7, 28.5 (x3), 25.6, 17.3 ppm.
4.1.7 tert-Butyl (trans-2-(4-aminophenyl)cyclopropyl) carbamate (6)
To a solution of compound 5 (0.14 g, 0.66 mmol, 1.0 eq) in THF: H2O 1:1 (1.8 mL) were added K2CO3 (64 mg, 0.46 mmol, 0.7
eq) and Pd/C (10%, 3.7 mg). A solution of Na2HPO2 (0.27 g, 2.5 mmol, 3.8 eq) in H2O (0.50 mL) was added to the mixture under
o
H2 atmosphere. The mixture was stirred at 60 C for 1.5h and, after completion, the reaction was quenched with H2O (20 mL) and
extracted with Et2O (3 x 20 mL). The organic phases were combined, dried over MgSO4, and evaporated under reduced pressure.
The residue was purified by column chromatography (EtOAc:petroleum ether 1:2) to afford 6 as a red oil (45 mg, 27%). Rf = 0.28
1
(EtOAc:petroleum ether 1:2). H NMR (400 MHz, CDCl3): δ = 6.93 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 8.4 Hz, 2H), 4.89 (bs, 1H),
3.59 (bs, 2H), 2.61 (m, 1H), 1.93 (m, 1H), 1.45 (s, 9H), 1.36 (m, 1H), 1.26 (m, 1H) ppm. 13C NMR (125 MHz, CDCl3): δ = 156.5,
144.6, 130.8, 127.8 (x2), 115.3 (x2), 79.6, 32.1, 29.8, 28.5 (x3), 15.8 ppm.
4.1.8 tert-Butyl (trans-2-(4-(2-(4-benzoylbenzamido)pent-4-ynamido)phenyl)cyclopropyl) carbamate (7)
In a 10 mL round bottom flask equipped with a stirring bar, compound 3 (68 mg, 0.2 mmol, 1.0 eq) and 6 (58 mg, 0.2 mmol, 1.1
eq) were dissolved in DCM (3.0 mL). EDC (49 mg, 0.3 mmol, 1.2 eq) was added at 0 oC and the mixture was left stirring at room
temperature overnight. The solvent was then evaporated and the residue was dissolved in EtOAc (5.0 mL), washed with 5% citric
acid (3 x 5 mL), NaHCO3 (3 x 5 mL), brine (1 x 5 mL). The organic layer was evaporated, dried over MgSO4 and evaporated under
reduced pressure. The residue was further purified by flash column chromatography (EtOAc:petroleum ether 1:3) to afford 7 as a
1
red oil (14 mg, 12%). Rf = 0.44 (EtOAc:petroleum ether 1:2). H NMR (500 MHz, CDCl3): δ = 8.54 (bs, 1H), 7.94 (d, J = 8.1 Hz,
2H), 7.87 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 7.4 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.51-7.48 (m, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.40 (d,
J = 7.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 4.96-4.90 (m, 1H), 4.88 (bs, 1H), 3.03-2.99 (m, 1H), 2.86-2.81 (m, 2H), 2.67 (m, 1H),
2.18-2.17 (m, 1H), 1.50 (m, 1H), 1.44 (s, 9H), 1.42 (m, 1H) ppm.
4.1.9 N-(1-((4-trans-(2-aminocyclopropyl)phenyl)amino)-1-oxopent-4-yn-2-yl)-4-benzoylbenzamide hydrochloride (8)
To a solution of 7 (14 mg, 0.03 mmol) in THF (1.0 mL) was added HCl 4N in dioxane (0.80 mL) at 0 oC and the mixture was left
stirring at room temperature overnight. The solvent was then evaporated and the residue was triturated with THF (2 x 1 mL) and
diethyl ether (1 x 1 mL) and then lyophilized to afford 8 as a brown solid (3.6 mg, 30%).1H NMR (400 MHz, CD3OD): δ = 8.04 (d,

J = 8.1 Hz, 2H), 7.86 (d, J = 8.1 Hz, 2H), 7.80 (d, J = 7.4 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.59-7.53 (m, 2H), 7.33-7.32 (m, 2H),
7.14 (d, J = 8.4 Hz, 2H), 4.75-4.74 (m, 1H), 2.91-2.87 (m, 2H), 2.85-2.80 (m, 1H), 2.45-2.42 (m, 1H), 2.36-2.35 (m, 1H), 1.50-1.45
(m, 1H), 1.38-1.34 (m, 1H) ppm. 13C NMR (100 MHz, CD3OD): δ = 196.4, 175.9, 174.7, 140.7, 138.6, 135.3, 134.2, 131.1, 130.9,
129.7 (x2), 129.2 (x2), 128.7 (x2), 127.8 (x2), 123.9 (x2), 121.8 (x2), 80.2, 72.5, 55.1, 31.9, 22.2, 22.1, 13.7. HRMS: C28H26O3N3
expected mass [M+H]+ 452.19687, mass found 452.19653.
4.1.10 tert-Butyl (trans-2-(4-(4-vinylbenzamido)phenyl) cyclopropyl)carbamate (9), tert-butyl (trans-2-(4-(pent-4-
ynamido)phenyl)cyclopropyl)carbamate (10)
Example: synthesis of 10. 4-Ethynylbenzoic acid (74 mg, 0.44 mmol, 1.2 eq), EDCI (0.10 g, 0.53 mmol, 1.4 eq), HOBt (82 mg,
0.53 mmol, 1.4 eq) and TEA (0.20 mL, 1.4 mmol, 3.8 eq) were added sequentially to a solution of 6 (94.2 mg, 0.38 mmol, 1.0 eq) in
dry DMF (2.8 mL). The resulting mixture was then stirred for 25h at room temperature and, after completion of the reaction,
quenched with NaHCO3 saturated solution (25 mL). The aqueous solution was extracted with EtOAc (4 x 20 mL), washed with
KHSO4 solution 0.1N (2 x 10 mL), NaHCO3 saturated solution (1 x 10 mL) and brine (1 x 10 mL), dried over anhydrous Na2SO4
and finally concentrated under vacuum. The crude was then purified by column chromatography on silica gel eluting with a mixture
EtOAc:hexane 22:78 to afford 10 as a pink solid (80 mg, 75%). Rf = 0.15 (EtOAc:n-hexane 1:2). 1H NMR (400 MHz, CDCl3): δ =
7.83-7.85 (d, J = 7.6 Hz, 2H), 7.59 (bs, 1H), 7.61-7.63 (d, J = 7.2 Hz, 2H), 7.53-7.55 (d, J = 7.2 Hz, 2H), 7.16-7.18 (d, J = 7.2 Hz,
2H), 4.80-4.89 (bs, 1H), 3.41 (s, 1H), 2.71-2.72 (m, 1H), 2.05-2.06 (m, 1H), 1.48 (s, 9H), 1.15-1.19 (m, 2H) ppm.
4.1.11 tert-Butyl (trans-2-(4-(pent-4-enamido)phenyl) cyclopropyl)carbamate (11)
Pent-4-enoyl chloride (0.047 mL, 0.43 mmol, 1.1 eq) was slowly added at 0 °C to a solution of 6 (90 mg, 0.39 mmol, 1.0 eq) and
TEA (0.07 mL, 0.50 mmol, 1.3 eq.) in dry DCM (16 mL). The resulting mixture was stirred at room temperature for 1.5h.
Afterwards, the reaction mixture was quenched with 10 mL of NaHCO3 saturated solution and extracted with EtOAc (4 x 10 mL).
The organic layer was washed with KHSO4 solution 0.1M (2 x 5 mL), and brine (2 x 5 mL), dried over anhydrous Na2SO4 and
concentrated in vacuum. The residue was purified by column chromatography on silica gel eluting with a mixture EtOAc:hexane
1
1:3 to afford 11 as a pink solid (56 mg, 76%). Rf = 0.15 (EtOAc:n-hexane 1:2). H NMR (400 MHz, CDCl3): δ = 7.40-7.42 (d, J =
8.4 Hz, 2H), 7.10-7.13 (m, 3H), 5.86-5.94 (m, 1H), 5.06-5.16 (m, 2H), 4.81-4.88 (bs, 1H), 2.67-2.69 (m, 1H), 2.41-2.43 (m, 4H),
2.02-2.04 (m, 1H), 1.47 (s, 9H), 1.12-1.14 (m, 2H) ppm.
4.1.12
N-(4-(trans-2-aminocyclopropyl)phenyl)-4-vinyl
benzamide
hydrochloride
(12),
N-(4-(trans-2-amino
cyclopropyl)phenyl)pent-4-enamide (13), N-(4-(trans-2-aminocyclopropyl)phenyl)pent-4-ynamide hydrochloride (14)
1
The same deprotection procedure was followed as for the synthesis of compound 8. 12: H NMR (400 MHz, (CD3)2SO): δ =
10.23 (bs, 1H), 8.43 (s, 3H), 7.94-7.96 (d, J = 7.6 Hz, 2H), 7.71-7.73 (d, J = 7.6 Hz, 2H), 7.62-7.64 (d, J = 7.6 Hz, 2H), 7.13-7.16
(d, J = 8.4 Hz, 2H), 6.79-6.86 (m, Jtrans = 17.6 Hz, Jcis = 12 Hz, 1H), 5.97-6.02 (d, Jtrans = 17.2 Hz, 1H), 5.39-5.42 (d, Jcis = 11.2 Hz,
1H), 2.77-2.79 (m, 1H), 2.31-2.33 (m, 1H), 1.33-1.38 (m, 1H), 1.16-1.24 (m, 1H) ppm. 13C NMR (100 MHz, (CD3)2SO): δ = 164.9,
140.1, 137.5, 135.9, 134.4, 133.9, 128.1(x2), 126.4(x2), 126.0(x2), 120.4(x2), 116.5, 30.5, 20.4, 13.2 ppm. HRMS: C18H19ON2
expected mass [M+H]+ 279.14919, mass found 279.14923.
13: 1H NMR (400 MHz, (CD3)2SO): δ = 9.92 (bs, 1H), 8.13-8.41 (m, 3H), 7.50-7.52 (d, J = 8.4 Hz, 2H), 7.06-7.08 (d, J = 8.4
Hz, 2H), 5.80-5.90 (m, 1H), 5.04-5.08 (d, Jtrans = 16 Hz, 1H), 4.97-4.99 (d, Jcis = 10 Hz, 1H), 2.71-2.75 (m, 1H), 2.37-2.41 (m, 2H),
2.30-2.35 (m, 2H), 2.23-2.25 (m, 1H), 1.29-1.33 (m, 1H), 1.12-1.18 (m, 1H) ppm. 13C NMR (100 MHz, (CD3)2SO): δ = 170.4,
137.7, 137.6, 133.6, 126.5(x2), 119.0(x2), 115.2, 35.4, 30.4, 29.1, 20.3, 13.1 ppm. HRMS: C14H19ON2 expected mass [M+H]+
231.14919, mass found 231.14922.
1
14: H NMR (400 MHz, (CD3)2SO): δ = 10.00 (bs, 1H), 8.41 (s, 3H), 7.52-7.54 (d, J = 8.4 Hz, 2H), 7.07-7.10 (d, J = 8.8 Hz,
2H), 2.80 (s, 1H), 2.73-2.77 (m, J = 5.8 Hz, 1H), 2.43-2.46 (m, 2H), 2.25-2.30 (m, 1H), 1.32-1.37 (m, J = 7.5 Hz, 1H), 1.13-1.18
(m, J = 7.0 Hz, 1H) ppm. 13C NMR (100 MHz, (CD3)2SO): δ = 169.2, 137.5, 133.8, 126.5(x2), 119.0(x2), 83.7, 71.5, 35.1, 30.4,
20.3, 14.1, 13.1 ppm. HRMS: C14H17ON2 expected mass [M+H]+ 229.13354, mass found 229.13354.
4.1.13 10-Acetyl-10H-phenoxazine-3,7-diyl diacetate (15)
1
The synthesis of Amplex Red reagent was performed as described in literature.57 Yield 69%. H NMR (500 MHz, CDCl3): δ =
7.46 (m, 2H), 6.91-6.88 (m, 4H), 2.33 (s, 3H), 2.30 (s, 6H) ppm. 13C NMR (125 MHz, CDCl3): δ = 169.2 (x2), 151.2, 149.0 (x2),
127.0, 125.5, 116.9, 111.0, 23.1, 21.2 (x2) ppm.
4.1.14 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) (16)
The synthetic procedure afforded a white solid (64%). 1H NMR (500 MHz, (CD3)2SO): δ = 9.7 (bs, 2H), 7.33 (m, 2H), 6.53 (m,
4H), 2.16 (s, 3H) ppm. 13C NMR (125 MHz, (CD3)2SO): δ = 169.0, 156.1, 151.0, 125.8, 121.0, 22.6 ppm.
4.2. LSD1 inhibition studies
4.2.1. General

LSD1 (KDM1A) (lysine (K)-specific demethylase 1A) or AOF2 was purchased from BPS Bioscience (Catalog No 50097).
Monomethylated histone peptide H3K4N(CH3) was purchased from Pepscan and Horseradish Peroxidase (HRP) from Pierce
(Catalog No 31490). The reagents for buffer preparation were purchased from Merck, Netherlands. Reactions were conducted in
black 96-well flat bottom microplates (Corning® Costar®, Corning Incorporated, NY). The fluorescence measurements were
carried out in a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, USA) and the gain setting of the instrument was
adjusted to 70. GraphPad Prism 5.0, GraphPad Software, Inc. GraphPad was used for the determination of kinetic values and of the
half maximal inhibitory concentration (IC50). Non-linear regression was used for data fitting.
4.2.2. LSD1 assay
The assay buffer consisted of Tris-HCl 50 mM, pH 8.0 containing 0.05 mg/mL Bovine Serum Albumine (BSA). Shortly before
use, LSD1 was diluted 500 times with LSD1 assay buffer. 35 μL of the diluted enzyme were used per well in the assay.
Monomethylated histone peptide H3K4N(CH3) was dissolved in the assay buffer to afford 2 mM stock solution. After proper
dilutions of this stock, 5 µL of the substrate were used per well in the assay. Horseradish Peroxidase (HRP, 300 U/mg) was diluted
to 10 U/mL using the LSD1 assay buffer. A stock solution of 10 mM Amplex Red in DMSO was prepared, protected from light and
used quickly. A solution of HRP-Amplex Red was prepared shortly before use, stored on ice and protected from light. 30 µL of the
stock solution of Amplex Red and 180 µL of the solution of HRP were added in 2.790 mL of LSD1 buffer. The addition of 50 μL of
this solution to the reaction wells is enough to use the H2O2 formed during the demethylation reaction for the oxidation of Amplex
Red.
The assay was based on a procedure described previously.58 The experiment was performed in triplicate. The inhibitors’ stock
solutions were prepared using the LSD1 assay buffer. 10 μL of each concentration of the inhibitor were added in the inhibitor wells.
10 μL of the solvent used to dissolve the inhibitor were added in the background and positive control wells. 50 μL of HRP-Amplex
Red solution were added in all wells. 35 μL of the diluted LSD1 were added in the positive and inhibitor wells and the contents
were mixed thoroughly. The plate was covered and incubated at room temperature for 15 min. 5 μL of a 30 µM solution of the
substrate were added in all wells. The plate was shaken and the fluorescence was monitored for 30 minutes at λem= 460 nm and λex=
390 nm. The slope of the linear part was calculated and the value of the background wells was subtracted from itself and the values
of the positive control and inhibitor wells. Non-linear regression was used to fit the data to the log(inhibitor) vs. response curve.
4.2.3. Testing for developer interference
10 μL of of appropriate stock solutions of the inhibitor were added in the inhibitor wells to afford final concentrations of 3-50
µM. 10 μL of the solvent of the inhibitor were added in the negative and positive control wells. 50 μL of HRP-Amplex Red Solution
were added in all wells. 35 μL of LSD1 buffer were added in all wells and the contents were mixed thoroughly. The plate was
covered and incubated at room temperature for 15 min. 5 μL of buffer were added in the negative control and 5 μL of 80 µM
solution of H2O2 were added in the inhibitor and positive control wells. The plate was shaken and the fluorescence was measured at
λem= 460 nm and λex= 390 nm. No interference was observed.
4.2.4. Kinetic studies-Kitz-Wilson analysis
LSD1 (41 ng) was incubated with four different concentrations of each compound for 0, 5, 10 and 15 min in presence of HRP-
Amplex Red solution. Positive and negative control wells were also set up. The substrate was then added at a final concentration of
1.5 µM and the enzyme activity was monitored as described above. The experiments were performed in triplicate. The slope of the
linear part was calculated and the value of the background wells was subtracted from itself and the values of the positive control and
inhibitor wells. A time-dependent inhibition mechanism was observed in all cases. The natural logarithm (ln) values of the
remaining enzyme activity for each inhibitor concentration were plotted versus the respective incubation time. The slope of the
resulting linear graphs corresponded to the reaction rates. Finally, the 1/rate values were plotted against 1/[inhibitor] to generate
linear graphs, fitting to the following equation: 1/rate=slope.(1/[inhibitor])+ki (ki=inactivation rate constant). The affinity rate
constant was calculated from the following equation: Ki= slope.ki. For all the compounds the Ki values ranged between 3-5 µM and
the ki between 0.3-0.4 min-1 indicating the very fast binding of the TCP-containing molecules.
4.3. Labeling studies
4.3.1. General
Human cervical cancer cells were used as a source of LSD1 activity (HeLa S3, ATCC® CCL-2.2™, USA). Dulbecco’s Modified
Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were purchased from Life Technologies. The antibodies used against LSD1
were purchased from ThermoFischer scientific (Catalog No PA5-17361 for the identification of intracellular LSD1 and Catalog No
PA5-23307 for the commercially available human recombinant LSD1). Fermentas PageRuler™ Prestained Protein Ladder was used
as a ladder during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The loading buffer (4x) consists of
20% of 0.2 M Tris-HCl pH 6.8, 8.9% of SDS, 40% of glycerol, 10% of 0.05 M EDTA, 0.09% of bromophenol blue, 21% of
deionized H2O. Gels were stained with the coomassie based gel stain InstantBlue™ (Expedeon Ltd, Harston, Cambridgeshire, UK).
Coomassie Protein Assay Reagent (950 mL) from Thermo Scientific was used for the Bradford assay and the absorbance was
measured on a SPECTROstar Omega–UV/Vis absorbance spectrophotometer microplate reader from BMG Labtech.
Chemiluminescence imaging was performed in G:BOX from Syngene under no light and no filter.

4.3.2. ‘Click’ reactions
10 µL of 10 mM solution of probe 8 in DMSO, 20 µL of azide-PEG3-biotin (5 mM in water), 40 µL of premixed catalyst
solution (20 µL TABTA 20 mM in H2O, 20 µL CuSO4 5 mM in water), 20 µL of sodium acorbate (0.1 M in H2O) and 10 µL of
DMSO were incubated at 32 oC for 5h. A blank reaction using 10 µL of DMSO was also set up. LC-MS revealed 50% conversion to
the desired clicked-probe 17. C46H57N9O8S expected mass [M+H]+ 896.41, mass found 896.41.
The same conditions were applied for the ‘click’ reaction using compound 2 (90% conversion, HRMS: C38H49N7O9S expected
mass [M+H]+ 780.33852, mass found 780.33706) and 14 (78% conversion, HRMS: C32H48N8O6S [M+H]+ 673.34948, pre-clicked
probe 18).
4.3.3. Labeling of human recombinant LSD1
1.25 µg of LSD1 (0.25 mg/mL, 5 µL) were incubated with 5, 10, 20 and 40 µM (final concentration) of pre-clicked probes 17
and 18 for 10 min (50 µL reaction in assay buffer). A blank reaction with no probe was also set up using the equivalent amount of
blank ‘click’ reaction conditions (see above). In case of probe 17 the samples were then irradiated at 365 nm for 5 min. For protein
detection methods via the anti-LSD1 antibody or HRP-conjugated Streptavidin, see Supplementary Information.
4.3.4. Labeling of endogenous LSD1 in Hela nuclear extracts
o
HeLa cells were grown in DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin, and maintained at 37 C
and 5% CO2 to afford approximately 68x105 cells. Hela nuclear extraction was performed as described previously.59 12.5, 25, 50
and 100 µM of probe 17 were incubated with the protein extracts (3.0 mg/mL in 100 µL assay buffer) for 10 min at room
temperature followed by 5 min irradiation at 365 nm. A blank reaction with no probe was also set up using the equivalent amount of
blank ‘click’ reaction conditions. In samples with 0, 12.5 and 25 µM, small molecules were removed by centrifuge in Viva spin
columns (Sartorius, 5 kDa membrane cut) at 4000 g for 12 min (x3). Samples were recollected by centrifuge at 3000 g for 2 min.
Samples containing 50 and 100 µM of the probe were treated with DMF (400 µL) and the proteins were left precipitating overnight
o
at 4 oC. Next morning, samples were centrifuged at 5200 rcf for 30 min at 4 C (x3, pellets resuspended with ice-cold MeOH). The
final protein pellets were resuspended in 10% SDS (150 µL). Further protein detection was performed as described in the
Supplementary Information. Same experimental conditions were applied for labeling of nuclear extracts with probe 18, where 0,
12.5, 25, 50 and 100 µM were incubated with 3 mg/mL of protein extracts for 15 min at room temperature. Samples were then
o
treated with DMF (400 µL) and the proteins were left precipitating overnight at 4 C. Detection was performed as described for
probe 17.
4.3.5. Control reactions
Regarding human recombinant LSD1, 1.25 µg of purified enzyme (0.25 mg/mL, 5 µL) in 41 µL of Tris 50 mM pH 8 were
incubated with probe 17 (final concentration 40 µM) for 5 min under UV irradiation at 365 nm (positive control). The equivalent
amount of blank ‘click’ reaction conditions (see above) were used as negative control. For heat-denaturation control (ΔT)
experiment, the same amount of LSD1 was pre-heated at 96 oC for 15 min and subsequently incubated with the probe as described
for positive control. To check that the probe targets the FAD, the same amount of LSD1 was incubated with pre-clicked compound
2 (final concentration 40 µM) for 5 min under UV irradiation at 365 nm. The same experiments were repeated for probe 18 (final
concentration 10 µM, no UV irradiation) and pre-clicked compound 2 was used at a final concentration of 10 µM. Detection was
performed as described above.
Nuclear extracts (3 mg/mL) were incubated with 100 µM of pre-clicked compound 2 or probe 17. The control reactions were
performed as described above for human recombinant LSD1. Proteins were precipitated with DMF (400 µL) overnight. Further
treatment and detection were performed as mentioned above for labeling of nuclear extracts.
4.3.6. Treatment with LSD1 inhibitors
Regarding human recombinant LSD1, 0.6 µg of purified enzyme (0.25 mg/mL, 2.5 µL) in 38.5 µL of Tris 50 mM pH 8 were
incubated with 12 (10 mM, 5 µL), TCP (15 mM, 5 µL) or DMSO (5 µL, positive control) for 30 min at room temperature. Pobe 17
(final concentration 40 µM) was then added in all samples followed by UV irradiation at 365 nm for 2 min. The same experiment
was repeated using pre-clicked probe 18 (final concentration 10 µM) incubated for 2 min after treatment with 10 mM of compound
12. Detection was performed as described above using longer exposure times for luminescence detection (1h).
HeLa nuclear extracts (3 mg/mL in LSD1 buffer) were incubated with 1.5 mM of TCP/1 mM of 12/ equivalent amount of
DMSO for 30 min at room temperature followed by treatment with 100 µM of probe 17 and 2 min UV irradiation or 50 µM probe
18 and 2 min incubation at room temperature. Ablank reaction was also set up. Further treatment and detection were performed as
mentioned above for labeling of nuclear extracts.
Acknowledgments
This work was financially supported by a VIDI grant (723.012.005) from the Netherlands Organization for Scientific Research
and an ERC starting grant (309782) from the European Union to F. J. D. Funds from PRIN 2016 (prot. 20152TE5PK) and IIT-

Sapienza Project grants are also acknowledged by A. M. PRIN 2012 (prot. 2012CTAYSY) and AIRC-TRIDEO 2015 (Id.17515) are
also acknowledged by D. R. We acknowledge the COST actions ‘biomimetic radical chemistry’ CM1201 and ‘epigenetic chemical
biology’ CM-1406.
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Graphical Abstract
.
Towards the development of activity-based
probes for detection of Lysine-Specific
Demethylase-1 activity
Leave this area blank for abstract info.
Maria E. Ourailidoua , Alessia Lenocib , Dante Rotilib, Antonello Maib,c,* and Frank J. Dekkera,*
aDepartment of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University
of Groningen, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands
bDepartment of Drug Chemistry and Technologies, ‘Sapienza’ University, P.le A. Moro 5, 00185 Rome, Italy
cPasteur Institute, Cenci Bolognetti Foundation, ‘Sapienza’ University, P.le A. Moro 5, 00185 Rome, Italy

Highlights

Lysine-specific demethylase 1 (LSD1) can be
covalently labelled using tranylcypromine (TCP)
based molecules.


LSD1 labeling is specific for non-denatured
enzymes.
Further investigation of the inhibition
mechanism of TCP based inhibitors for LSD1 is
needed.