Comparative analysis of small molecules and histone substrate analogues as LSD1 lysine demethylase inhibitors

Article abstract of DOI:10.1021/ja909996p

LSD1 is a flavin-dependent histone demethylase that oxidatively removes methyl groups from Lys-4 of histone H3. LSD1 belongs to the amine oxidase enzyme superfamily which utilize molecular oxygen to transform amines to iminjes that are hydrolytically cleaved to formaldehyde. In prior studies, it has been shown that monoamine oxidase inhibitory scaffolds such as propargylamines and cyclopropylamines can serve as mechanism-based inactivators of LSD1. Propargylamine-histone H3 peptide analogues are potent LSD1 inhibitors, whereas small molecule antidepressant MAO acetylenic inhibitors like pargyline do not inhibit LSD1. In contrast, the small molecule MAO cyclopropylamine inhibitor tranylcypromine is a timedependent LSD1 inhibitor but exo-cyclopropylamine- peptide substrate analogue is not. To provide further insight into small molecule versus peptide relationships in LSD1 inhibition, herein we further our analysis of warheads in peptide scaffolds to include the chlorovinyl, endo-cyclopropylamine, and hydrazinefunctionalities as LSD1 inactivators. We find that chlorovinyl-H3 is a mechanism-based LSD1 inactivator whereas encto-cyclopropylamine-H3 does not show time-dependent inactivation. The hydrazine-H3 was shown to be the most potent LSD1 suicide inhibitor yet reported, more than 20-fold more efficient in inhibiting demethylation than propargylamine-H3 derivatives. We re-explored MAO antidepressant agent phenelzine (phenethylhydrazine), previously reported to be a weak LSD1 inhibitor, and found that it is far more potent than previously appreciated. We show that phenelzine can block histone H3K4Me demethylation in cells, validating it as a pharmacologic tool and potential lead structure for anticancer therapy.

Full text of DOI:10.1021/ja909996p

Published on Web 02/11/2010
Comparative Analysis of Small Molecules and Histone
Substrate Analogues as LSD1 Lysine Demethylase Inhibitors
Jeffrey C. Culhane,† Dongqing Wang,‡ Paul M. Yen,‡,§ and Philip A. Cole*,†
Department of Pharmacology and Molecular Sciences, Johns Hopkins UniVersity School of
Medicine, Baltimore, Maryland 21205, Department of Medicine, Johns Hopkins BayView
Medical Center, Baltimore, Maryland 21224, and CardioVascular and Metabolic Diseases
Program, Duke-NUS Graduate Medical School, Singapore 169857
Received November 25, 2009; E-mail: pcole@jhmi.edu
Abstract: LSD1 is a flavin-dependent histone demethylase that oxidatively removes methyl groups from
Lys-4 of histone H3. LSD1 belongs to the amine oxidase enzyme superfamily which utilize molecular oxygen
to transform amines to imines that are hydrolytically cleaved to formaldehyde. In prior studies, it has been
shown that monoamine oxidase inhibitory scaffolds such as propargylamines and cyclopropylamines can
serve as mechanism-based inactivators of LSD1. Propargylamine-histone H3 peptide analogues are potent
LSD1 inhibitors, whereas small molecule antidepressant MAO acetylenic inhibitors like pargyline do not
inhibit LSD1. In contrast, the small molecule MAO cyclopropylamine inhibitor tranylcypromine is a time-
dependent LSD1 inhibitor but exo-cyclopropylamine-peptide substrate analogue is not. To provide further
insight into small molecule versus peptide relationships in LSD1 inhibition, herein we further our analysis
of warheads in peptide scaffolds to include the chlorovinyl, endo-cyclopropylamine, and hydrazine-
functionalities as LSD1 inactivators. We find that chlorovinyl-H3 is a mechanism-based LSD1 inactivator
whereas endo-cyclopropylamine-H3 does not show time-dependent inactivation. The hydrazine-H3 was
shown to be the most potent LSD1 suicide inhibitor yet reported, more than 20-fold more efficient in inhibiting
demethylation than propargylamine-H3 derivatives. We re-explored MAO antidepressant agent phenelzine
(phenethylhydrazine), previously reported to be a weak LSD1 inhibitor, and found that it is far more potent
than previously appreciated. We show that phenelzine can block histone H3K4Me demethylation in cells,
validating it as a pharmacologic tool and potential lead structure for anticancer therapy.
Post-translational modification (PTM) of histones on lysines
regulates gene expression by remodeling chromatin and is a
central focus of epigenetic studies.1,2 Among the many PTMs
that reversibly modify chromatin, lysine methylation provides
for a rich array of biological readouts.3,4 For example, methy-
lation of Lys-4 of histone H3 is a mark of gene activation,
whereas Lys-9 methylation is more often associated with gene
silencing. The relatively recent discovery of histone lysine
demethylases has helped round out our understanding of the
factors that control methyl-Lys stability.5,6 Included among the
histone demethylases are the flavin-dependent enzymes LSD1
and LSD2 and the iron-dependent Jmj catalysts.5–7
removal of methyl groups, specifically from Lys-4 of histone
H3.5,8 The catalytic cycle of methyl removal produces a
molecule each of formaldehyde and H2O2 while consuming O2
(Scheme 1).5,8 Dictated by the inherent limitations of the flavin
chemistry, LSD1 can only function in the removal of the mono
and dimethyl species of Lys-4.5 LSD1 functions as a transcrip-
tional repressor and is a component of various transcriptional
corepressor complexes that often include HDAC1/2 and
CoREST.9-12 In addition to processing histone proteins, LSD1
can demethylate H3-tail peptides, requiring at least 15 amino
acid residues for efficient demethylation.5,13 Inhibitors of LSD1
would be expected, in general, to reactivate gene expression of
silenced genes, which might have utility in the treatment of
cancer and other diseases.14,15 Such compounds could for
example be synergistic with HDAC inhibitors.14,15
LSD1 is a member of the amine oxidase superfamily and
utilizes a noncovalently bound FAD cofactor in the oxidative
† Johns Hopkins University School of Medicine.
‡ Johns Hopkins Bayview Medical Center.
(8) Binda, C.; Mattevi, A.; Edmonson, D. E. J. Biol. Chem. 2002, 277,
23973–23976.
§ Duke-NUS Graduate Medical School.
(1) Taverna, S. D.; Li, H.; Ruthenburg, A. J.; Allis, C. D.; Patel, D. J.
Nat. Struct. Mol. Biol. 2007, 14, 1025–1040.
(9) Hakimi, M. A.; Dong, Y.; Lane, W. S.; Speicher, D. W.; Shiekhattar,
R. J. Biol. Chem. 2003, 278, 7234–7239.
(2) Cole, P. A. Nat. Chem. Biol. 2008, 4, 590–597.
(10) Humphrey, G. W.; Wang, Y.; Russanova, V. R.; Hirai, T.; Qin, J.;
Nakatani, Y.; Howard, B. H. J. Biol. Chem. 2001, 276, 6817–6824.
(11) Shi, Y.; Sawada, J.; Sui, G.; Affar el, B.; Whetstine, J. R.; Lan, F.;
Ogawa, H.; Luke, M. P.; Nakatani, Y.; Shi, Y. Nature 2003, 422,
735–738.
(3) Martin, C.; Zhang, Y. Nat. ReV. Mol. Cell. Biol. 2005, 6, 838–849.
(4) Kouzarides, T. Cell 2007, 282, 15040–15047.
(5) Shi, Y.; Lan, F.; Matson, C.; Mulligan, P.; Whetstine, J. R.; Cole,
P. A.; Casero, R. A.; Shi, Y. Cell 2004, 119, 941–953.
(6) Tsukada, Y.; Fang, J.; Erdjument-Bromage, H.; Warren, M. E.;
Borchers, C. H.; Tempst, P.; Zhang, Y. Nature 2006, 439, 811–816.
(7) Karytinos, A.; Forneris, F.; Profumo, A.; Ciossani, G.; Battaglioli,
E.; Binda, C.; Mattevi, A. J. Biol. Chem. 2009, 284, 17775–17782.
(12) You, A.; Tong, J. K.; Grozinger, C. M.; Schreiber, S. L. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 1454–1458.
(13) Forneris, F.; Binda, C.; Vanoni, M. A.; Battaglioli, E.; Mattevi, A.
J. Biol. Chem. 2005, 280, 41360–41365.
9
3164 J. AM. CHEM. SOC. 2010, 132, 3164–3176
10.1021/ja909996p  2010 American Chemical Society

LSD1 Lysine Demethylase Inhibitors
A R T I C L E S
Scheme 1. Catalytic Cycle of Flavin Mediated Amine Oxidationa
a Oxidation of the amine to an iminium ion, with concurrent reduction of the flavin enables attack by a closely held water molecule. Collapse of the
hemiaminal to a carbonyl yields formaldehyde and amine. Reduced flavin is re-oxidized by molecular oxygen, generating a molecule of hydrogen peroxide.
The flavin-dependent amine oxidase family of enzymes has
been intensively studied over the past 50 years as clinical targets
for human diseases.16 In particular, the monoamine oxidases,
MAO-A and MAO-B, play key roles in the clearance of
neurotransmitters.16–18 Selective MAO-A/B suicide inactivators
have enjoyed decades of clinical success in the treatment of
major depression and neurodegenerative disorders such as
Parkinson’s disease.16–18 Since LSD1 and MAO-A/B share a
common mechanism for the oxidative cleavage of the unacti-
vated nitrogen-carbon bonds of their substrates, many of the
known MAO inactivators have been tested as LSD1 inhibitors.14
Tranylcypromine, a cyclopropylamine containing small mol-
ecule, has been characterized as an LSD1 inactivator through
biochemical, spectroscopic, and crystallographic procedures.14,19,20
The hydrazine-containing MAO inhibitor and antidepressant
phenelzine has also been reported to be a weak LSD1 inhibitor.14
On the other hand, pargyline, a propargylamine containing small
molecule initially suggested to be an LSD1 inhibitor, has failed
in subsequent studies to appreciably inactivate LSD1.13,14
Interestingly, the propargylamine functionality in the context
of a histone H3-21 peptide (1) has yielded potent time- and
concentration-dependent inactivation of LSD1 (Figure 1).21-23
Characterization of the inactivation by 1 and its N-methyl
analogue 2 by kinetic, spectroscopic, and crystallographic studies
have revealed key mechanistic and structural information about
the nature of inactivation and substrate recognition.21–23 In
particular, an X-ray crystal structure of LSD1 inactivated by 2
has provided a model for histone H3 substrate recognition.23
These studies have established that MAO inhibitor functional-
Figure 1. Structures of evaluated LSD1 inhibitors and inactivators.
(14) Lee, M. G.; Wynder, C.; Schmidt, D. M.; McCafferty, D. G.;
ities can target LSD1 but leave open the range of warheads and
the contextual relationship to substrate analogues that can inhibit
this enzyme. In this study, we investigate other known MAO-
A/B inactivator motifs for incorporation into histone H3-21
peptides in the search for increasingly potent inactivators of
LSD1. We compare and contrast the behavior of small molecules
versus peptide analogues containing inhibitory warheads. Im-
portantly, we identify the hydrazine motif as the most potent
LSD1 inhibitor functionality in the context of peptides and small
molecules.
Shiekhattar, R. Chem. Biol. 2006, 13, 563–567.
(15) Huang, Y.; Greene, E.; Stewart, T. M.; Goodwin, A. C.; Baylin, S. B.;
Woster, P. M.; Casero, R. A., Jr. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 8023–8028.
(16) Edmondson, D. E.; Mattevi, A.; Binda, C.; Li, M.; Huba´lek, F. Curr.
Med. Chem. 2004, 11, 1983–1993.
(17) Yu, P. H. Gen. Pharmacol. 1994, 25, 1527–1539.
(18) Goodman, W. K.; Charney, D. S. J. Clin. Psychiatry. 1985, 46, 6–24.
(19) Schmidt, D. M.; McCafferty, D. G. Biochemistry 2007, 46, 4408–
4416.
(20) Yang, M.; Culhane, J. C.; Szewczuk, L. M.; Jalili, P.; Ball, H. L.;
Machius, M.; Cole, P. A.; Yu, H. Biochemistry 2007, 46, 8058–8065.
(21) Culhane, J. C.; Szewczuk, L. M.; Liu, X.; Da, G.; Marmorstein, R.;
Cole, P. A. J. Am. Chem. Soc. 2006, 128, 4536–4537.
(22) Szewczuk, L. M.; Culhane, J. C.; Yang, M.; Majumdar, A.; Yu, H.;
Cole, P. A. Biochemistry 2007, 46, 6892–6902.
Results and Discussion
Chlorovinyl-H3 Peptide Analogue Synthesis and LSD1
Inhibition. The 3-chloroallylamine functionality has the ability
to inactivate flavin-dependent amine oxidases,24,25 and we
(23) Yang, M.; Culhane, J. C.; Szewczuk, L. M.; Gocke, C. B.; Brautigam,
C. A.; Tomchick, D. R.; Machius, M.; Cole, P. A.; Yu, H. Nat. Struct.
Mol. Biol. 2007, 14, 535–539.
9
J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010 3165

A R T I C L E S
Culhane et al.
Scheme 2. Synthesis of Peptide Inhibitors cis-3-Chloroallyl-Lys-4 H3-21 (3), trans-3-Chloroallyl-Lys-4 H3-21 (4) and Hydrazino-Lys-4 H3-21
(18)a
a Incorporation of Fmoc-hydroxynorleucine in the four position of H3-21 yields the resin bound peptide. Mesylation of the alcohol while on bead, followed
by trifluoroacetic acid cleavage and RP-HPLC purification provides mesyl-Lys-4 H3-21 peptide 5. Displacement of the mesylate by cis- or
trans-3-chloroallylamine or hydrazine yields the desired peptide inhibitors. Single letter amino acid abbreviations represent the H3-21 sequence.
therefore sought to generate H3-chlorovinyl analogues 3 and
4. For this purpose, we prepared cis-3-chloroallylamine hydro-
chloride from commercially available cis-1,3-dichloropropene
utilizing the Gabriel synthetic approach.25 The corresponding
trans-3-chloroallylamine hydrochloride was generated in a
similar fashion from commercially available rac-1,3-dichloro-
propene followed by selective precipitation.25 Peptide analogues
3 and 4 were synthesized utilizing our previously reported
strategy.21 Briefly, a 21 amino acid N-terminal histone H3 tail
peptide was constructed on solid support incorporating an oxa-
analogue of lysine, 6-hydroxynorleucine,21 at the fourth position.
Following mesylation of the alcohol, the peptide was cleaved
from the resin and universally deprotected, allowing for reverse
phase high performance liquid chromatography (RP-HPLC)
purification of the mesylate peptide (5). Displacement of the
mesylate with small molecule amines afforded the desired
peptides in 40-50% yield (Scheme 2).
Peptides 3 and 4 were assayed against recombinant GST-
LSD1 utilizing a horseradish peroxidase coupled assay for the
detection of H2O2 produced in the demethylase catalytic cycle.21
cis-3-Chloroallyl-Lys-4 H3-21 (3) and trans-3-chloroallyl-Lys-4
H3-21 (4) both displayed time- and concentration-dependent
inactivation of LSD1 with Ki(inact) ) 0.955 ( 0.15 and 0.763 (
0.12 µM, and kinact ) 0.545 ( 0.038 and 0.128 ( 0.0087 min-1,
respectively (Figure 2, Table 1). It has been hypothesized that
the chlorovinyl mechanism of inactivation of MAO is similar
to that of a propargylamine inactivator.24 We propose, in analogy
to previous studies on MAOs, that oxidation of 3 and 4 produces
R,ꢀ-unsaturated iminium electrophiles that are capable of
Michael addition with a nearby nucleophile. The presence of
the chloride as a leaving group allows for the generation of a
conjugated diamine linker (Scheme 3). The 4-fold difference
in the inactivation rate constants for the cis (3) versus trans (4)
isomer may be related to altered oxidation rates or follow-on
nucleophilic attack differences that stem from the steric and
spatial orientation of the chloride atom in the active site of the
enzyme. The efficiency of inactivation (kinact/Ki(inact)) of LSD1
by 3 is similar to that of the propargylamine compound 1 and
within 5-fold of the kinact/Ki(inact) of the more potent N-
methylpropargylamine peptide 2 (Figure 1, Table 1).
To further investigate the inactivation processes for 3 and 4,
we employed UV-visible spectroscopy to characterize possible
flavin adducts, as seen previously with compounds 1 and 2.21–23
LSD1-bound FAD has two characteristic absorption maxima
in the 350-550 nm range, attributed to the fully oxidized flavin
and the one electron reduced semiquinone form.26 As with 1,
treatment of LSD1 with inactivator 3 or 4 leads to the near
bleaching of the flavin spectrum. The cis isomer (3) results in
a newly formed maximum at 383 nm, while the trans isomer
(4) induces a new maximum at a wavelength less than 350 nm
(Figure 3). It is possible that these spectroscopic shifts cor-
respond to inhibitor-flavin adducts with different stereochemistry.
The FAD molecule of LSD1 is noncovalently bound. Inac-
tivator-FAD adducts can therefore be isolated from the protein
prior to analysis by MALDI-TOF mass spectrometry.20–23 Mass
spec analysis of denatured 3 or 4-inactivated LSD1 showed
peaks at m/z ) 3077, corresponding to the mass of the peptide
and the FAD following the loss of the chloride atom, as
proposed in Scheme 3 (Figure 4). Additionally, for both
inactivation reactions, a peak corresponding to H3-21 peptide
is noted at m/z ) 2255, this degradation product may be
(24) Rando, R. R.; Eigner, A. Mol. Pharmacol. 1997, 13, 1005–1013.
(25) Jeon, H. B.; Sayre, L. M. Biochem. Biophys. Res. Commun. 2003,
304, 788–794.
(26) Woo, J. C. G.; Silverman, R. B. Biochem. Biophys. Res. Commun.
1994, 202, 1574–1578.
9
3166 J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010

LSD1 Lysine Demethylase Inhibitors
A R T I C L E S
Figure 2. Time- and concentration-dependent inactivation of GST-LSD1 by cis-3-chloroallyl-Lys-4 H3-21 (3) and trans-3-chloroallyl-Lys-4 H3-21 (4). (A
and C) Steady-state progress curves obtained for the inactivation of LSD1 in the presence of concentrations of 3 ranging from 0 to 28.8 µM and concentrations
of 4 ranging from 0 to 27.2 µM. (B and D) Rate constants (kobs) for the time-dependent inactivation of LSD1 by 3 and 4 were extracted from the steady-state
data in (A) and (C) by single exponential fits and analyzed by the method of Kitz and Wilson to determine kinact and Ki(inact) values.
Table 1. Kinetics of Evaluated GST-LSD1 Inhibitor and Inactivator Peptides and Small Moleculesa
Inhibitor/Inactivator
Ki(inact) (µM)
k(inact) (min-1
)
k(inact)/Ki(inact) (µM-1min-1
)
Ki (µM)
propargyl-Lys-4 H3-21 (1)
0.775 ( 0.16
0.107 ( 0.057
0.955 ( 0.15
0.763 ( 0.12
-
0.304 ( 0.033
0.208 ( 0.068
0.545 ( 0.038
0.128 ( 0.0087
-
0.392 ( 0.0088
1.94 ( 0.34
0.571 ( 0.0062
0.168 ( 0.0018
-
-
-
-
-
N-methylpropargyl-Lys-4 H3-21 (2)
cis-3-chloroallyl-Lys-4 H3-21 (3)
trans-3-chloroallyl-Lys-4 H3-21 (4)
exo-cyclopropyl-Lys-4 H3-21 (6)
endo-cyclopropyl-Lys-4 H3-21 (7)
endo-dimethylcyclopropyl-Lys-4 H3-21 (8)
hydrazino-Lys-4 H3-21 (18)
tranylcypromine
2.70 ( 0.74
-
-
-
6.11 ( 0.86
24.2 ( 2.7
-
-
-
-
-
-
0.00435 ( 0.00086
357 ( 120
17.6 ( 2.8
0.247 ( 0.018
0.542 ( 0.094
0.955 ( 0.085
56.8 ( 0.82
0.00152 ( 0.000089
0.0543 ( 0.00077
phenelzine
a Data for compounds 1, 2, 6, and tranylcypromine have been obtained previously.20–23
Scheme 3. Proposed Mechanism of Inactivation of GST-LSD1 by cis-3-Chloroallyl-Lys-4 H3-21 (3)a
a Flavin mediated two electron oxidation of 3 yields an R,ꢀ-unsaturated iminium species. Pathway (a) Nucleophilic attack by the N5 of the reduced flavin
leads to an R-chloro eneamine intermediate. Subsequent loss of the chloride atom forms the conjugated diamine linked 3-FAD conjugate. Pathway (b)
Nucleophilic attack on the activated species by a water molecule leads to H3-21 peptide degradation product. Pathway (c) Nucleophilic attack on the
activated species by a nucleophile contained in the peptide leads to a cyclic peptide degradation product.
generated from an active site water molecule attacking the
oxidatively activated iminum species at the R carbon as shown
in Scheme 3, path b. Another potential degradation peak is also
noted at m/z ) 2290. The mass of the product corresponds to
the loss of HCl from the oxidized intermediates produced from
3 or 4. It is formally possible that after the activation of 3 and
9
J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010 3167

A R T I C L E S
Culhane et al.
Figure 3. Spectroscopic analysis of cis- or trans-3-chloroallyl-Lys-4 H3-
21 (3 and 4) treated GST-LSD1. UV/vis spectra of native LSD1 (blue)
shows the distinctive two maxima of the fully oxidized flavin and the one
electron reduced semiquinone. (A) LSD1 treated with 3 leads to a shift in
the absorbance to a new maximum at 383 nm. (B) LSD1 treated with 4
leads to a shift in the absorbance to a new maximum less than 350 nm.
Figure 4. MALDI-TOF analysis of GST-LSD1 incubated with cis- or trans-
3-chloroallyl-Lys-4 H3-21 (3 and 4). (A) Inactivator 3 is seen as a minor
peak, while a new signal corresponding to the predicted mass of a 3-FAD
adduct is now apparent. Additionally, a signal corresponding to the
hydrolysis of 3, forming H3-21 is noted. Possible intramolecular cyclization
of the activated peptide is seen as the major peak. (B) Inactivator 4 is seen
as a minor peak, while a new signal corresponding to the predicted mass
of a 4-FAD adduct is now apparent. A signal corresponding to the hydrolysis
of 4, forming H3-21, is noted. Possible intramolecular cyclization of the
activated peptide is seen as the major peak.
4 by LSD1 an intramolecular cyclization of the peptide thru
Michael addition, potentially within or outside the active site,
leads to the degradation of the inactivator, as shown in Scheme
3, path c. Consistent with these degradation mechanisms of the
inactivator, only a minor peak in the mass spectrum corre-
sponding to the mass of peptide 3 or 4 is observed after LSD1
treatment. Taken together, these studies support an inactivation
mechanism involving flavin attack on the conjugated imine as
proposed in Scheme 3. It is difficult to obtain precise partition
ratios, however, because of the challenge in separating and
quantifying the various enzymatic products by HPLC.
Endo-Cyclopropyl-H3 Synthesis and LSD1 Inhibition
Analysis. The small molecule tranylcypromine displays mod-
erately potent time- and concentration-dependent inactivation
of LSD1 (Table 1).20 In contrast, an H3-21 peptide that
incorporated an exo-cyclopropyl-Lys-4 functionality (6) was
found to be a reversible inhibitor (estimated Ki ) 2.70 ( 0.74
µM), but did not inactivate LSD1 (Figure 1, Table 1).22 It can
be rationalized that this result is due to steric clashes related to
the bulky exo-cyclopropyl group of 6. To explore this further,
we synthesized the corresponding endo-cyclopropylamine con-
taining peptides 7 and 8 which better mimic the structural
arrangements found in tranylcypromine (Figure 1). To that end,
an efficient synthesis of the Fmoc-endo-cyclopropyl-
Lys(Boc)-OH (9) and Fmoc-endo-dimethylcyclopropyl-Lys-
OH (10) monomers was designed for their direct incorporation
during solid phase peptide synthesis. While previous syntheses
exist for endo-cyclopropyl containing lysine residues,27 our route
maintains the proper four carbon chain between the peptide
backbone and the epsilon nitrogen of lysine.
Perbenzylation of commercially available L-Glu (11), selective
reduction of the side chain ester to the alcohol (12), and Swern
oxidation yields the amino aldehyde 13 in an overall 43% yield
(Scheme 4).28 Horner-Wadsworth-Emmons reaction of the
aldehyde 13 with t-butyl diethylphosphonoacetate provides the
R,ꢀ-unsaturated ester 1429 for cyclopropanation with diaz-
omethane yielding 15.30 The R,ꢀ-cyclopropyl t-butyl ester 15
can be selectively hydrolyzed under acidic conditions allowing
the acid 16 to undergo Curtius rearrangement to yield the tert-
butyl carbamate protected cyclopropylamine 17.31 Removal of
the benzyl esters can be accomplished under hydrogenation
conditions in a Parr shaker to provide the deprotected R-amino
acid. Treatment with Fmoc-OSu yields Fmoc-endo-cyclopropyl-
Lys(Boc)-OH (9). TFA treatment of 9 allows for the incor-
poration of two methyl groups by reductive amination, yielding
Fmoc-endo-dimethylcyclopropyl-Lys-OH (10) (Scheme 5).32
(28) Kokotos, G.; Padron, J. M.; Martin, T.; Gibbons, W. A.; Martin, V. S.
J. Org. Chem. 1998, 63, 3741–3744.
(29) Barrett, A. G. M.; Head, J.; Smith, M. L.; Stock, N. S.; White, A. J. P.;
Williams, D. J. J. Org. Chem. 1999, 64, 6005–6018.
(30) Tchilibon, S.; Kim, S. K.; Gao, Z. G.; Harris, B. A.; Blaustein, J. B.;
Gross, A. S.; Duong, H. T.; Melman, N.; Jacobson, K. A. Bioorg.
Med. Chem. 2004, 12, 2021–2034.
(31) Raju, B.; Anandan, S.; Gu, S.; Herradura, P.; O’Dowd, H.; Kim, B.;
Gomez, M.; Hackbarth, C.; Wu, C.; Wang, W.; Yuan, Z.; White, R.;
Trias, J.; Patel, D. V. Bioorg. Med. Chem. Lett. 2004, 14, 3103–3107.
(32) Arvidsson, L. E.; Johansson, A. M.; Hacksell, U.; Nilsson, J. L. G.;
Svensson, K.; Hjorth, S.; Magnusson, T.; Carlsson, A.; Lindberg, P.;
Andersson, B.; Sanchez, D.; Wikstrom, H.; Sundell, S. J. Med. Chem.
1988, 31, 92–99.
(27) Brandl, M.; Kozhushkov, S. I.; Loscha, K.; Kokoreva, O. V.; Yufit,
D. S.; Howard, J. A. K.; de Meijere, A. Synlett 2000, 12, 1741–1744.
9
3168 J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010

LSD1 Lysine Demethylase Inhibitors
A R T I C L E S
Scheme 4. Synthesis of the Amino Aldehyde (S)-Benzyl 2-(dibenzylamino)-5-oxopentanoate (13)
Scheme 5. Synthesis of Fmoc-endo-cyclopropyl-Lys(Boc)-OH (9) and Fmoc-endo-dimethylcyclopropyl-Lys-OH (10)
Fmoc amino acids 9 and 10 were used in standard Fmoc SPPS
to provide peptides 7 and 8.22
Hydazino Analogues as LSD1 Inhibitors. As mentioned,
phenelzine was previously shown to be a weak inhibitor of
LSD1, suggested to be 20-fold weaker than tranylcypromine.14
To analyze this further, we embarked on the synthesis of
hydrazine analogue 18. This was readily achieved by displace-
ment of the mesylate functionality with hydrazine akin to
generation of inactivators 3 and 4 (Scheme 2).21 Initial
enzymatic inhibition studies revealed compound 18 to be a
highly potent time-dependent inhibitor of LSD1. Limited
sensitivity of the 4-aminoantipyrine and 3,5-dichloro-2-hydroxy-
benzenesulfonic acid coupled assay21 prevented a precise
determination of the inhibitory parameters. We therefore utilized
a more sensitive Amplex Red33 coupled assay for the detection
of H2O2 produced during enzymatic turnover of substrate. We
determined that hydrazine analogue 18 had a Ki(inact) and k(inact)
In contrast to the results with propargylamine and chlorovinyl
derivatives, endo-cyclopropylamine containing peptides 7 and
8 failed to inactivate LSD1. Both peptides displayed a reversible
mode of inhibition with an estimated Ki ) 6.11 ( 0.86 µM
and 24.2 ( 2.7 µM, respectively, determined by Dixon analysis
assuming a competitive inhibitory model (Figure 5). Interest-
ingly, the dimethylated peptide (8) displayed lower potency than
the unmethylated analogue (7), although 8 was projected to be
the better model of the dimethyl-Lys-4 substrate. This result
suggests that the incorporation of the cyclopropyl moiety not
only as exo as in peptide 6,22 but also as endo effects the binding
of the inhibitor peptide enough to eliminate oxidative turnover
by LSD1. We suggest that the radical/cation stabilizing function
of the benzyl group, lacking in 7 and 8, plays a key effector
function in the tranylcypromine inactivation mechanism of
LSD1.
(33) Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. Anal.
Biochem. 1997, 253, 162–168.
9
J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010 3169

A R T I C L E S
Culhane et al.
Figure 5. Inhibition of GST-LSD1 by endo-cyclopropyl-Lys-4 H3-21 (7) and endo-dimethylcyclopropyl-Lys-4 H3-21 (8). (A and C) Steady-state progress
curves obtained for the inhibition of LSD1 in the presence of concentrations of 7 ranging from 0 to 49.6 µM and concentrations of 8 ranging from 0 to 720
µM. (B and D) Steady-state velocities obtained in (A) and (C) were used to determine the Ki for compounds 7 and 8 by Dixon analysis.
versus the amino functionality contributes to enhanced affinity,
and that the neutral rather than the positively charged species
preferentially binds to LSD1. Alternatively, the Ki(inact) of 18
may not correspond to its Kd but instead may be composed of
a series of complex rate constants.
Spectroscopic analysis of 18 inactivated LSD1 showed loss
of the visible maxima, consistent with flavin modification
(Figure 7A). The MALDI mass spectrum of the inactivated
mixture revealed a peak with m/z ) 3024, consistent with the
formation of a peptide-FAD adduct with concurrent loss of N2
(Figure 7B). In accordance with prior proposals for phenelzine
inactivation of MAO, we suggest an LSD1 inactivation mech-
anism that initially involves a two electron oxidation to form
the corresponding diazene (Scheme 6, path a). We propose that
after reoxidation of the FAD by molecular oxygen, a two
electron oxidation of the diazene yields the diazonium species,
an excellent leaving group. Attack from the N5 of the reduced
flavin leads to the inactivator-FAD adduct with lose of N2
(Scheme 6 path b).34,35 Interestingly, the mass spectrum also
shows evidence of two peptide degradation pathways. The first
correlates to an aldehyde containing peptide at m/z ) 2253.
This product could potentially stem from nonenzymatic hy-
drolysis of a hydrazone that can be produced during the initial
oxidation of the inhibitor to the diazene (Scheme 6, path d). A
second degradation correlates to the loss of N2H2 from the
oxidatively activated diazene peptide (m/z ) 2237). This could
Figure 6. Time- and concentration-dependent inactivation of GST-LSD1
potentially be produced through the abstraction of the beta
proton and lose of N2 yielding an olefin (Scheme 6, path c), or
through an internal cyclization of the peptide as similarly
proposed previously in the case of the chlorovinyl inactivators
(Scheme 3). Quantification of the relative product ratios in the
LSD1 reaction with 18 is difficult because of the challenge of
separating and detecting these chemical species by HPLC. We
cannot also not rule out the possibility that the LSD1 inactivation
mechanism related to 18 also involves some covalent enzyme
modification reactions.
by hydrazino-Lys-4 H3-21 (18). (A) Steady-state progress curves obtained
for the inactivation of LSD1 in the presence of concentrations of 18 ranging
from 0.1 to 0.4 µM. (B) Rate constants (kobs) for the time-dependent
inactivation of LSD1 by 18 were extracted from the steady-state data in
(A) by single exponential fits and analyzed by the method of Kitz and Wilson
to determine kinact and Ki(inact) values.
of 4.35 ( 0.86 nM and 0.247 ( 0.018 min-1, respectively
(Figure 6). This makes hydrazino-Lys-4 H3-21 (18) ap-
proximately 25-fold more potent than 2, the previous best in
class LSD1 inactivator which contains the N-methylpropargy-
lamine motif (Table 1). The significantly lower Ki(inact) of 18
compared with that of 1 and 2 was unexpected and may reflect
a higher affinity for the encounter complex between 18 and
LSD1. If so, this may suggest that the lower pKa of the hydrazine
(34) Clineschmidt, B. V.; Horita, A. Biochem. Pharmacol. 1969, 18, 1011–
1020.
(35) Yu, P. H.; Tipton, K. F. Biochem. Pharmacol. 1989, 38, 4245–4251.
9
3170 J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010

LSD1 Lysine Demethylase Inhibitors
A R T I C L E S
that the assay methods were quite distinct. Moreover, the
tranylcypromine LSD1 inhibition parameters measured previ-
ously by us20 were in close agreement with those of Schmidt
and McCafferty.19
Given the relative in vitro inhibitory potency of phenelzine
toward recombinant LSD1, we considered that phenelzine might
also block LSD1 in live cells. To examine the effects of
phenelzine as a demethylase inhibitor in cells, we explored the
effects of phenelzine on a thyroid hormone (T3) inhibited TSHR
luciferase reporter transfected in cells.38 As shown, LSD1
inhibition increases luciferase activity both in the absence and
presence of T3 but maintains negative regulation by T3 (Figure
11). Assessment of the methylation status of Lys-4 of histone
H3 by ChIP (chromatin immunoprecipation) in response to
phenelzine, revealed that mono and dimethylation of the TSHR
reporter region was enhanced by phenelzine, whereas the
trimethylation level was unaffected (Figure 12). These findings
suggest that mono- and dimethylation of Lys-4 H3 may enhance
basal transcription of TSHR promoter in the absence or presence
of T3. These results correlate with that expected for LSD1
inhibition (as opposed to a trimethyl-Lys demethylase) and
establish the pharmacologic value of phenelzine as an epigenetic
probe.
Summary
In this study, we have designed, synthesized, and examined
several novel H3 tail peptide analogues containing classical
monoamine oxidase warhead groups as LSD1 inhibitors. The
chlorovinyl-containing inhibitors are comparable in potency to
alkynyl inhibitors described previously, whereas the hydrazine
containing H3 peptide is the most potent suicide inactivator
known for LSD1. Consistent with this finding, the hydrazine
containing MAO inhibitor phenelzine is the most potent small
molecule LSD1 inhibitor that has emerged from our studies.
Its potency is such that the pharmacology of phenelzine, which
is clearly active against MAOs, is likely enhancing Lys-4 H3
methylation in its therapeutically useful range. It could for
example synergize with HDAC and DNA methyltransferase
inhibitors to induce the re-expression of silenced tumor sup-
pressor genes.39,40
In contrast to the peptide and small molecule hydrazines, there
is discordance in inactivation potential of propargyl and
cyclopropyl inhibitors of LSD1. Whereas propargylamine pep-
tide inhibitors are efficient LSD1 inhibitors, peptide cyclopropyl
compounds are not. This contrasts with the pargyline and
tranylcypromine effects on LSD1. Taken together, these studies
reveal a complex mosaic in molecular recognition among LSD1,
its substrates, and inhibitory compounds.
Figure 7. Spectroscopic and MALDI-TOF analysis of hydrazino-Lys-4
H3-21 (18) treated GST-LSD1. (A) UV/vis spectra of native LSD1 (blue)
shows the distinctive two maxima of the fully oxidized flavin and the one
electron reduced semiquinone. LSD1 treated with 18 (red) leads to the
bleaching of the flavin spectra with no new maximum recorded. (B) In the
MALDI-TOF analysis, 18 is seen as a minor peak, while a major peak
corresponding to the predicted mass of an 18-FAD adduct is now evident.
Additionally, a signal corresponding to the hydrolysis of 18, forming the
aldehyde containing peptide is noted along with a possible degradation to
the olefin or intramolecular cyclization.
The impressive inhibitory characteristics with hydrazino-
Lys-4 H3-21 (18) led us to reinvestigate the inhibitory charac-
teristics of phenelzine for LSD1. Remarkably, we found that
phenelzine showed a Ki(inact) of 17.6 ( 2.8 µM and a k(inact) of
0.955 ( 0.085 min-1 (Figure 8). To rule out that the perceived
LSD1 inhibition was somehow related to the interfering action
of phenelzine on peroxide detection, we performed the following
additional experiments. We demonstrated that inactivation of
LSD1 was greater when pretreated with phenelzine, in the
absence of horseradish peroxidase, followed by assay (Figure
9A). We showed that additional horseradish peroxidase failed
to relieve the LSD1 inhibition (Figure 9B). Finally, we
determined in a direct assay using mass spectrometry that
phenelzine-treated LSD1 was unable to induce loss of a methyl
group from an H3-21 dimethyl-Lys substrate peptide (Figure
10). While considerably less potent than hydrazino-Lys-4 H3-
21 (18), phenelzine is approximately 35-fold more efficient as
an LSD1 inactivator than tranylcypromine in our hands, and
appears to be comparable to a newly described class of
tranylcypromine analogues.36 The inactivation efficiency of
phenelzine toward LSD1 appears comparable to, if not greater
than, its inhibitory effect versus MAOs.37 Although we cannot
account for the inhibitory differences regarding tranylcypromine
and phenelzine between our work and a prior study,14 we note
Experimental Section
General. All synthetic reactions were carried out under an inert
argon atmosphere using standard techniques. Solvents were pur-
chased from Aldrich as anhydrous and used as is. NMR spectra
were recorded on a Varian 400 MHz spectrometer. MALDI-TOF
spectra were recorded on a Applied Biosystems Voyager DE-STR
mass spectrometer. ESI-TOF spectra were recorded on an Applied
Biosystems Sciex instrument.
cis-3-Chloroallyl-Lys-4 H3-21 (3). Lyophilized mesyl-Lys-4 H3-
21 (5) (5.0 mg, 2.1 µmol) was dissolved in 500 µL of 1:1 H2O/
(38) Wang, D.; Xia, X.; Liu, Y.; Oetting, A.; Walker, R. L.; Zhu, Y.;
Meltzer, P.; Cole, P. A.; Shi, Y. B.; Yen, P. M. Mol. Endocrinol.
2009, 23, 600–609.
(36) Ueda, R.; Suzuki, T.; Mino, K.; Tsumoto, H.; Nakagawa, H.;
Hasegawa, M.; Sasaki, R.; Mizukami, T.; Miyata, N. J. Am. Chem.
Soc. 2009, 131, 17536–17537.
(39) Jones, P. A.; Baylin, S. B. Cell 2007, 128, 683–692.
(40) Minucci, S.; Pelicci, P. G. Nat. ReV. Cancer 2006, 6, 38–51.
(37) Patek, D. R.; Hellerman, L. J. Biol. Chem. 1974, 249, 2373–2380.
9
J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010 3171

A R T I C L E S
Culhane et al.
Scheme 6. Proposed Mechanism of Inactivation of GST-LSD1 by Hydrazino-Lys-4 H3-21 (18)a
a Pathway (a), two electron oxidation of 18 by the flavin yields the diazene. A second oxidation to the primary diazonium species produces a powerful
electrophilic site at the alpha carbon. Pathway (b), nucleophilic attack by the flavin N5 yields inactivated LSD1 as an 18-FAD adduct. Pathway (c), abstraction
of the beta proton leads to the olefin through lose of N2. Pathway (d), the competing oxidation leads to the formation of the hydrazone which can non-
enzymatically hydrolyze to the aldehyde containing peptide oxo-Lys-4 H3-21.
Figure 9. GST-LSD1 inactivation by phenelzine. (A) LSD1 inactivation
is more pronounced when preincubated with phenelzine. (B) LSD1
inactivation is independent of the horseradish peroxidase enzyme concentration.
Figure 8. Time- and concentration-dependent inactivation of GST-LSD1
by the small molecule MAO inactivator phenelzine. (A) Steady-state
CH3CN. cis-3-Chloroallylamine hydrochloride (82 mg, 640 µmol)
progress curves obtained for the inactivation of LSD1 in the presence of
in 500 µL of 1:1 H2O/CH3CN was added to the solution followed
concentrations of phenelzine ranging from 15.6 to 250 µM. (B) Rate
by freshly distilled triethylamine (120 µL, 860 µmol). The reaction
constants (kobs) for the time-dependent inactivation of LSD1 by phenelzine
rotated 70 h at 25 °C. The crude reaction mixture was diluted to
15 mL with H2O, acidified to pH 2 with TFA, and lyophilized to
an oil. The oil was diluted to 3 mL with H2O and injected onto a
were extracted from the steady-state data in A by single exponential fits
and analyzed by the method of Kitz and Wilson to determine kinact and
Ki(inact) values.
9
3172 J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010

LSD1 Lysine Demethylase Inhibitors
A R T I C L E S
Figure 10. MALDI-TOF assay demonstrating the inactivation of GST-
LSD1 by phenelzine. (A) LSD1 preincubated with phenelzine prior to
exposure to dimethyl-Lys-4 H3-21 substrate fails to appreciably demethylate.
(B) LSD1 efficiently demethylates dimethyl-Lys-4 H3-21 substrate yielding
monomethyl Lys-4 H3-21 and Lys-4 H3-21.
prep scale column for RP-HPLC purification. Analysis by MALDI-
TOF showed an expected/observed m/z ) 2328.31.
Figure 11. T3 and phenelzine effects on TSHR promoter activity and Lys-4
H3 methylation. R-23 cells were treated 24 h with no hormone/phenelzine
(control), 0.1 µM T3, 10 µM phenelzine, or both. The cell lysates were
prepared and luciferase activities were measured as described in Experi-
mental Section. Shown are the mean of triplicate samples ( SD normalized
to control as 100%. Similar findings were found in two other experiments.
Data were analyzed by ANOVA and * ) p < 0.05 and ** ) p < 0.01
difference between indicated samples and EtOH control samples.
trans-3-Chloroallyl-Lys-4 H3-21 (4). Lyophilized mesyl-Lys-4
H3-21 (5) (5.0 mg, 2.1 µmol) was dissolved in 500 µL of 1:1 H2O/
CH3CN. trans-3-chloroallylamine hydrochloride (82 mg, 640 µmol)
in 500 µL of 1:1 H2O/CH3CN was added to the solution followed
by freshly distilled triethylamine (120 µL, 860 µmol). The reaction
rotated 70 h at 25 °C. The crude reaction mixture was diluted to
25 mL with H2O, acidified to pH 2 with TFA, and lyophilized to
an oil. The oil was diluted to 3 mL with H2O and injected onto a
prep scale column for RP-HPLC purification. Analysis by MALDI-
TOF showed an expected/observed m/z ) 2328.31.
Mesyl-Lys-4 H3-21 (5). The primary alcohol of resin bound
peptide was treated with 20 equivalents of mesyl chloride in the
presence of 40 equivalents of triethylamine in tetrahydrofuran for
20 h at room temperature. Universal deprotection and cleavage of
the peptide from the Wang resin, in the presence of the mesylate,
was accomplished by treating the resin bound peptide with Reagent
K (95:5 trifluoroacetic acid/H2O in the presence of phenol,
ethanedithiol, and thioanisole) for 4 h at room temperature.
Precipitation of the peptide with diethyl ether followed by lyo-
philization yielded crude peptide as an off-white solid that was
purified by prep scale RP-HPLC. Analysis by MALDI-TOF showed
an expected/observed m/z ) 2333.28.
Figure 12. T3 and phenelzine regulation of methylation of Lys-4 H3. ChIP
assay was performed as described in the Experimental section using
antibodies against mono-, di- and trimethylated H3K4. Similar findings were
observed in two other experiments. The Y axis shows relative H3K4
methylation based upon changes in cycle threshold number above back-
ground after each treatment. Data were analyzed by ANOVA and * ) p <
0.05 difference between indicated samples and EtOH control samples.
endo-Cyclopropyl-Lys-4 H3-21 (7). Standard Fmoc solid phase
peptide synthesis technique was utilized to assemble the endo-
cyclopropyl-Lys-4 H3-21 peptide. The endo-cyclopropyl-Lys-4
residue was inserted as the Fmoc monomer 9. Universal deprotec-
tion and cleavage of the peptide from the Wang resin was
accomplished with Reagent K (95:5 trifluoroacetic acid: H2O in
the presence of phenol, ethanedithiol, and thioanisole) for 5 h at
25 °C. Precipitation of the peptide with diethyl ether followed by
lyophilization yielded crude peptide as an off-white solid that was
purified by prep scale RP-HPLC. Analysis by MALDI-TOF showed
an expected/observed m/z ) 2266.31.
the endo-dimethylcyclopropyl-Lys-4 H3-21 peptide. The endo-
dimethylcyclopropyl-Lys-4 residue was inserted as the Fmoc
monomer 10. Universal deprotection and cleavage of the peptide
from the Wang resin was accomplished with Reagent K (95:5
trifluoroacetic acid/H2O in the presence of phenol, ethanedithiol,
and thioanisole) for 5 h at 25 °C. Precipitation of the peptide with
endo-Dimethylcyclopropyl-Lys-4 H3-21 (8). Standard Fmoc
solid phase peptide synthesis technique was utilized to assemble
9
J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010 3173

A R T I C L E S
Culhane et al.
diethyl ether followed by lyophilization yielded crude peptide as
an off-white solid that was purified by prep scale RP-HPLC.
Analysis by MALDI-TOF showed an expected/observed m/z )
2294.35.
before being filtered through a pad of Celite. The filtrate was dried
over MgSO4, filtered, and concentrated in Vacuo to an oil. The crude
product was purified by silica gel column chromatography in
petroleum ether/diethyl ether (90:10-50:50) to yield 12 g (80%)
of 12 as a clear viscous oil.28 1H (CD3Cl3, 400 MHz): δ 7.45-7.21
(m, 15H); 5.21 (q, J ) 31.5 and 12 Hz, 2H); 3.91 (d, J ) 14 Hz,
2H); 3.51 (d, J ) 14 Hz, 2H); 3.48 (q, J ) 5.8 Hz, 2H); 3.38 (t, J
) 7.6 Hz, 1H); 1.81 (m, 2H); 1.71 (m, 2H) 1.48 (m, 2H).
(S)-Benzyl 2-(dibenzylamino)-5-oxopentanoate (13). 28 Oxalyl
chloride (2.25 mL, 25.8 mmol) in 140 mL anhydrous dichlo-
romethane at -78 °C had DMSO (3.65 mL, 51.5 mmol) added
dropwise and stirred 25 min under an argon atmosphere. Alcohol
12 (5.2 g, 12.9 mmol) in 50 mL of anhydrous dichloromethane
was added dropwise to this solution and stirred an additional 30
min. Triethylamine (8.6 mL, 61.8 mmol) was added to the reaction
and stirred 30 min at -78 °C, triethylamine (8.6 mL, 61.8 mmol)
was again added to the reaction and allowed to warm to 0 °C over
30 min. 100 mL of H2O was added to quench the reaction while
warming to 25 °C. The organic phase was separated and the aqueous
phase was extracted 2 × 100 mL with dichloromethane. The pooled
organics were washed 1 × 75 mL with saturated sodium bicarbonate
and saturated brine, dried over MgSO4, filtered, and concentrated
in Vacuo to a golden oil. Residual solvent was removed by high
vacuum over 2 h. The product aldehyde was used without further
purification yielding 5.0 g (97%) of 13 as an oil.28 1H (CD3Cl3,
400 MHz): δ 9.59 (s, 1H); 7.43-7.23 (m, 15H); 5.23 (q, J ) 27.6
and 12 Hz, 2H); 3.87 (d, J ) 13.6 Hz, 2H); 3.50 (d, J ) 13.6 Hz,
2H); 3.35 (t, J ) 7.8 Hz, 1H); 2.53 (m, 1H); 2.42 (m, 1H); 2.03
(m, 2H); 1.55 (m, 2H).
Fmoc-endo-cyclopropyl-Lys(Boc)-OH (9). The benzylated
amino acid 17 (600 mg, 1.1 mmol) in 10 mL of absolute ethanol
had 100 mg of palladium on carbon (10 wt % wet) added to it and
the suspension degassed with argon. Hydrogenation, using a Parr
shaker apparatus, was performed for 15 h under 55 psi of hydrogen
gas. Following the hydrogenation, 10 mL of H2O was added to the
suspension to dissolve the free amino acid. The suspension was
filtered through a plug of Celite and washed with H2O and ethanol.
The solvent was removed in Vacuo to yield a white solid which
was then suspended in 10 mL of 1:1 H2O/Acetone. Potassium
carbonate (684 mg, 4.95 mmol) and Fmoc-succinimidyl carbonate
(668 mg, 1.98 mmol) were added and the reaction was stirred at
25 °C for 15 h. The reaction was concentrated in Vacuo to an off-
white solid and purified by RP-HPLC on a prep scale column using
a H2O/Acetonitrile gradient with 0.05% formic acid to yield 106
1
mg (20%) of 9 as a white powder following lyophilization. H
(CD3Cl3, 400 MHz): δ 7.75 (d, J ) 7.2 Hz, 2H); 7.58 (d, J ) 7.2
Hz, 1H); 7.38 (t, J ) 7.2 Hz, 2H); 7.29 (t, J ) 7.2 Hz, 2H); 5.67
(d, 1H); 4.95 (d, 1H); 4.67 (m, 1H); 4.37 (t, 2H); 4.21 (d, 1H);
2.46 (m, 1H); 2.20 (m, 1H); 2.06 (m, 1H); 1.79 (m, 2H); 0.94 (m,
1H); 0.62 (m, 1H); 0.48 (m, 1H). HRMS: expected: 481.23 [M+H],
observed: 503.2125 [M + Na].
Fmoc-endo-dimethylcyclopropyl-Lys-OH · TFA (10).32 Fmoc
amino acid 9 (40 mg, 0.083 mmol) in 1 mL of dichloromethane
was cooled to 0 °C while stirring. One mL of trifluoroacetic acid
was added and stirred for 15 min before being allowed to warm to
25 °C over 45 min. The reaction was concentrated in Vacuo to an
oil and residual solvent was removed by high vacuum over 2 h.
The amine was dissolved in 1.5 mL of acetonitrile followed by the
addition of formaldehyde (37% w/w Aq, 12.5 mg, 0.415 mmol).
Sodium cyanoborohydride (15.6 mg, 0.249 mmol) was added as
one portion and stirred 5 min. Glacial acetic acid was added
dropwise to keep the pH 4-5. The reaction stirred 15 h before
being concentrated in Vacuo to an oil and purified by RP-HPLC
on a prep scale column using a H2O: Acetonitrile gradient with
0.05% trifluoroacetic acid to yield 30 mg (70%) of 10 as a white
(S,E)-7-Benzyl 1-tert-butyl 6-(dibenzylamino)hept-2-enedioate
29
(14).
tert-butyl diethylphosphonoacetate (4.4 mL, 18.7 mmol)
was added dropwise to a stirring suspension of sodium hydride
(60% dispersion in oil, 717 mg, 18.7 mmol) in 50 mL of anhydrous
tetrahydrofuran at 0 °C under an argon atmosphere. The reaction
is allowed to warm to 25 °C over 30 min before being cooled to
-10 °C with an ice/acetone bath. Aldehyde 13 (5.0 g, 12.5 mmol)
in 50 mL of anhydrous tetrahydrofuran was added dropwise to the
solution and stirred 30 min. After the solvent was removed in Vacuo,
the oil was partitioned in 100 mL of H2O and diethyl ether. The
aqueous phase was extracted 1 × 50 mL with diethyl ether. The
pooled organics were washed 1 × 50 mL with saturated sodium
bicarbonate and saturated brine, dried over MgSO4, filtered, and
concentrated in Vacuo to a golden oil. The crude product was
purified by silica gel column chromatography in petroleum ether/
diethyl ether (99:1-90:10) to yield 5.3 g (85%) of 14 as a slightly
yellow oil.291H (CD3Cl3, 400 MHz): δ 7.44-7.20 (m, 15H); 6.70
(dt, J ) 15.2 and 6.8 Hz, 1H); 5.58 (dt, J ) 15.2 and 1.2 Hz); 5.21
(q, J ) 28.7 and 12 Hz, 2H); 3.88 (d, J ) 14 Hz, 2H); 3.50 (d, J
) 14 Hz, 2H); 3.34 (t, J ) 7.6 Hz, 1H); 2.32 (m, 1H); 2.05 (m,
1H); 1.86 (m, 2H); 1.46 (s, 9H). HRMS: expected: 500.27 [M+H],
observed: 500.2799 [M+H].
1
powder following lyophilization. H (CD3Cl3, 400 MHz): δ 7.72
(d, 2H); 7.55 (t, 2H); 7.35 (t, 2H); 7.26 (t, 2H); 5.93 (d, 1H); 4.40
(m, 1H); 4.30 (d, 2H); 4.14 (t, 1H); 2.79 (bs, 6H); 2.29 (m, 1H);
2.00 (m, 1H); 1.80 (m,1H); 1.50 (m, 2H); 1.22 (m, 2H); 0.68 (m,
1H). HRMS: expected: 409.20, observed: 409.2118 [M + H].
(S)-Dibenzyl 2-(dibenzylamino)pentanedioate (11). 28 Glutamic
acid (12.4 g, 84.2 mmol), potassium carbonate (46.5 g, 337 mmol),
and potassium hydroxide (9.5 g, 168 mmol) in 125 mL of H2O
was brought to reflux while stirring. Benzyl bromide (50.0 mL,
421 mmol) was added dropwise over 30 min and allowed to reflux
an additional 30 min. The cooled reaction was extracted with diethyl
ether 3 × 75 mL. The pooled organics were washed with saturated
brine 2 × 50 mL. The organic phase was washed 2 × 75 mL with
brine and dried over MgSO4, filtered, and concentrated in Vacuo
to an oil. The crude product was purified by silica gel column
chromatography in hexane/ethyl acetate (99:1-80:20) to yield 17 g
(56%) of 11 as a clear viscous oil.28 1H (CD3Cl3, 400 MHz): δ
7.41-7.19 (m, 20H); 5.21 (q, J ) 30.4 and 12.4 Hz, 2H); 4.98 (q,
J ) 12.4 and 5.2 Hz, 2H); 3.88 (d, J ) 14 Hz, 2H); 3.49 (d, J )
14 Hz, 2H); 3.41 (t, J ) 7.6 Hz, 1H); 2.51 (m, 1H); 2.35 (m, 1H);
2.06 (q, J ) 7.2 Hz, 2H).
tert-Butyl 2-((S)-4-(benzyloxy)-3-(dibenzylamino)-4-oxobu-
30
tyl)cyclopropane carboxylate (15). Unsaturated tert-butyl ester
14 (5.25 g, 10.5 mmol) in 2:1 anhydrous dichloromethane/
anhydrous diethyl ether was cooled to 0 °C while stirring under an
argon atmosphere. Palladium(II) acetate (17.7 mg, 0.079 mmol)
was added as one portion and stirred 5 min. Diazomethane
(estimated at 0.37 M in diethyl ether, 100 mL, 37 mmol) (see below
for diazomethane generation procedure) was added dropwise over
35 min via liquid addition funnel. The reaction was allowed to warm
to 25 °C while stirring overnight. The reaction was filtered through
a plug of Celite and concentrated in Vacuo to a golden oil. Residual
solvent was removed by high vacuum over 3 h. The cyclopropyl
tert-butyl ester was used without further purification yielding 5.3 g
28
(S)-Benzyl 2-(dibenzylamino)-5-hydroxypentanoate (12).
The benzyl protected amino acid 11 (19 g, 37.4 mmol) in 200 mL
of anhydrous tetrahydrofuran was cooled to -10 °C with an ice/
acetone bath while stirring under an argon atmosphere. DIBAL (1
M in toluene, 112 mL, 112 mmol) was added dropwise over 40
min. Following the addition, the reaction was warmed to 0 °C with
an ice water bath and stirred 100 min. The reaction was quenched
by the addition of 80 mL of H2O and stirred an addition 20 min
1
(98%) of 15 as an oil. H (CD3Cl3, 400 MHz): 2 predominant
rotamers present δ 7.42-7.20 (m, 15H); 5.20 (q, J ) 33.2 and 12
Hz, 2H); 3.89 (d, J ) 14 Hz, 2H); 3.50 (d, J ) 14 Hz, 2H); 3.35
(t, J ) 7.6 Hz, 1H); 1.83 (m, 2H); 1.42 (s, 9H); 1.29 (m, 1H); 1.17
9
3174 J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010

LSD1 Lysine Demethylase Inhibitors
A R T I C L E S
(m, 2H); 1.12 (m, 1H); 0.96 (m, 1H); 0.47 (m, 1H). HRMS:
expected 514.29 [M + H], observed 514.2955 [M + H].
Diazomethane. Diazomethane was generated according to
Aldrich technical bulletin AL-180 in an Aldrich mini diazald
apparatus. Diazald (5.0 g, 23 mmol) in 45 mL of diethyl ether was
added dropwise over 20 min to a 65 °C stirring solution of
potassium hydroxide (2.5 g, 44.6 mmol) in 4 mL of H2O, 8 mL of
diethyl ether, and 14 mL of 2-(2-ethoxyethoxy)ethanol. The
intensely yellow-colored distillate is collected and stored at -78
°C in a brown bottle and used within 2 h.
2-((S)-4-(Benzyloxy)-3-(dibenzylamino)-4-oxobutyl)-cyclo-
propanecarboxylic acid (16). Cyclopropyl tert-butyl ester 15 (5.3
g, 10.3 mmol) in 100 mL of dichloromethane was cooled to 0 °C
while stirring. 100 mL of trifluoroacetic acid was added and stirred
for 15 min before being allowed to warm to 25 °C over 45 min.
The solvent was removed in Vacuo, and the oil partitioned in 100
mL of H2O and dichloromethane. The aqueous phase was extracted
2 × 25 mL with dichloromethane. The pooled organics were washed
1 × 50 mL with saturated brine, dried over MgSO4, filtered, and
concentrated in Vacuo to an oil. Residual solvent was removed by
high vacuum over 2 h. The carboxylic acid was used without further
purification yielding 4.7 g (99%) of 16 as an oil. 1H (CD3Cl3, 400
MHz): δ 7.43-7.20 (m, 15H); 5.21 (q, J ) 28.4 and 12 Hz, 2H);
3.90 (d, J ) 14 Hz, 2H); 3.54 (d, J ) 14 Hz, 2H); 3.36 (t, J ) 7.2
Hz, 1H); 1.84 (m, 2H); 1.43 (m, 1H); 1.32 (m, 1H); 1.25 (m, 2H);
1.10 (m, 1H); 0.62 (m, 1H). HRMS: expected 458.23 [M + H],
observed 458.2326 [M + H].
25 000× g for 30 min. The clarified lysate from 1 L of culture was
double loaded (0.5 mL/min) onto a 5 mL glutathione sepharose 4
fast flow column (GE Healthcare) that was pre-equilibrated with
lysis buffer. The column was then washed with 75 mL of lysis
buffer and eluted with 5 × 5 mL fractions of lysis buffer containing
50 mM reduced glutathione (Sigma). GST-LSD1 containing frac-
tions were pooled and dialyzed against 3 × 1 L changes of lysis
buffer containing 1 mM ꢀ-mercaptoethanol instead of 10 mM DTT.
The dialyzed protein was concentrated to 1-2 mL and further
purified by size exclusion chromatography using sephacryl S100
high resolution media (GE Healthcare, 1.5 × 90 cm column). The
protein was eluted with lysis buffer containing 1 mM ꢀ-mercap-
toethanol instead of 10 mM DTT at a flow rate of 0.25 mL/min.
GST-LSD1 containing fractions were pooled, concentrated, ali-
quoted, and stored at -80 °C. Final protein concentration was
determined by Bradford assay using BSA as the standard. Purifica-
tion of GST-LSD1 by this procedure yielded approximately 1 mg
of protein (>90% pure) per 1 L of culture.
LSD1 Activity Assays. Initial velocity measurements were
performed using a peroxidase-coupled assay, which monitors
hydrogen peroxide production as previously described.21 The time
courses of the reaction were measured under aerobic conditions
using a Beckman Instruments DU series 600 spectrophotometer
equipped with a thermostatted cell holder (T ) 25 °C). The 150
µL reactions were initiated by the addition of 50 µL of buffered
substrate (dimethyl-Lys-4 H3-21) solution to reaction mixtures (100
µL) consisting of 50 mM HEPES buffer (pH 7.5), 0.1 mM
4-aminoantipyrine, 1 mM 3,5-dichloro-2-hydroxybenzenesulfonic
acid, 0.76 µM horseradish peroxidase (Worthington Biochemical
Corporation), and 185 nM GST-LSD1. Absorbance changes were
monitored at 515 nm, and an extinction coefficient of 26 000 M-1
cm-1 was used to calculate product formation. Under these
conditions, GST-LSD1 displayed at kcat of 4.5 ( 0.1 min-1 and a
Km for dimethyl-Lys-4 H3-21 of 21 ( 2 µM. A secondary assay
was necessary in the case of inactivator 18. In this case, the 150
µL reactions were initiated by the addition of 50 µL of buffered
substrate (dimethyl-Lys-4 H3-21) solution to reaction mixtures (100
µL) consisting of 50 mM HEPES buffer (pH 7.5), 0.1 mM Amplex
Red, 0.76 µM horseradish peroxidase, and 25 nM LSD1. Absor-
bance changes were monitored at 571 nm, and an extinction
coefficient of 52 000 M-1 cm-1 was used to calculate product
formation. Under these conditions, our GST-LSD1 displayed at kcat
of 3.5 ( 0.2 min-1 and a Km for dimethyl-Lys-4 H3-21 of 20 ( 3
µM.
(2S)-Benzyl 4-(2-(tert-butoxycarbonylamino)cyclopropyl)-
2-(dibenzylamino) butanoate (17).31 Acid 16 (4.7 g, 10.3 mmol)
in 100 mL of anhydrous toluene was cooled to 0 °C while stirring
under an argon atmosphere. Triethylamine (4.25 mL, 30.9 mmol)
and diphenylphosphorylazide (4.54 mL, 21.0 mmol) were added
and the reaction warmed to 25 °C over 3 h. The reaction was
washed 3 × 50 mL with H2O, 1 × 25 mL with saturated brine,
dried over MgSO4, filtered, and concentrated in Vacuo. Residual
solvent was removed by high vacuum over 4 h. The azide was
dissolved in 100 mL of anhydrous tert-butanol while stirring under
an argon atmosphere and heated to reflux for 18 h. The reaction
was cooled to 25 °C and concentrated in Vacuo to a golden oil.
The oil was partitioned between 100 mL of H2O and dichlo-
romethane. The organic phase was washed 1 × 25 mL with
saturated sodium bicarbonate and saturated brine, dried over
MgSO4, filtered, and concentrated in Vacuo. The crude product was
purified by silica gel column chromatography in petroleum ether:
diethyl ether (95:5-60:40) to yield 3.6 g (66%) of 17 as a clear
Inhibition Studies. Inhibitors were tested by using the peroxi-
dase-coupled assay described above. In these experiments, assays
were initiated by the addition of buffered substrate and the inhibitor
simultaneously. Final substrate concentrations were 60 µM (3 ×
Km when examining 7), 100 µM (5 × Km when examining 8), 240
µM (12 × Km when examining 3, 4, and phenelzine) or 600 µM
(30 × Km when examining 18). With the exception of the evaluation
of 18, GST-LSD1 was present at 185 nM. Inactivator peptide 18
required a more sensitive assay and was analyzed using Amplex
Red as the horseradish peroxidase cosubstrate rather than 4-ami-
noantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid as
noted previously, LSD1 was present in these assays at 25 nM.
Progress curves obtained in the presence of inactivators 3, 4, 18,
and phenelzine were fit to the following single exponential for slow-
binding inhibitors which assumes a steady-state velocity of zero:41
1
oil. H (CD3Cl3, 400 MHz): δ 7.44-7.20 (m, 15H); 5.21 (q, J )
32.8 and 12 Hz, 2H); 3.89 (d, J ) 14 Hz, 2H); 3.49 (d, J ) 14 Hz,
2H); 3.38 (t, J ) 7.6 Hz, 1H); 2.13 (m, 1H); 1.84 (m, 2H); 1.44 (s,
9H); 1.38 (m, 1H); 1.18 (m, 1H); 0.55 (m, 1H); 0.49 (m, 1H); 0.37
(m, 1H). HRMS: expected 529.30 [M + H], observed 529.3064
[M + H].
Hydrazino-Lys-4 H3-21 (18). Lyophilized mesyl-Lys-4 H3-21
(5) (5.0 mg, 2.1 µmol) was dissolved in 750 µL of 1:1 H2O/CH3CN.
Hydrazine monohydrate (57 µL, 860 µmol) was added to the
solution and rotated 70 h at 25 °C. The crude reaction mixture was
diluted to 3 mL with H2O, acidified to pH 2 with TFA, and injected
onto a prep scale column for RP-HPLC purification. Analysis by
MALDI-TOF showed an expected/observed m/z ) 2269.33.
Enzyme Expression and Purification. LSD1 (aa 171-852)
subcloned into the pGEX-6P-1 vector (GE Healthcare) was over-
expressed in E. coli BL21-CodonPlus(DE3)-RIPL cells (Strat-
Product ) V0(1 - e-kt)/k + offset
(1)
agene).22 Cells were grown to an OD600
of 1.8 in CircleGrow
nm
Media (Q-Biogene) at 37 °C then induced with 1 mM final IPTG
and grown for 20 h at 16 °C. Cell pellets were harvested by
centrifugation at 5000× g for 15 min and resuspended in ice cold
lysis buffer (280 mM NaCl, 5.4 mM KCl, 20 mM Na2HPO4, 3.6
mM KH2PO4, 1 mM EDTA, 10 mM DTT, 10% glycerol, pH 7.4).
The cells were then lysed via double pass on a French press
(16 000-18 000 psi), and the lysates clarified by centrifugation at
The kobs values were then analyzed by the method of Kitz and
Wilson to yield kinact and Ki(inact). The following equation was used
(41) Copeland, R. A. Enzymes: A Practical Introduction to Structure,
Mechanism, and Data Analysis, 2nd ed.; Wiley-VCH: New York,
2000.
9
J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010 3175

A R T I C L E S
Culhane et al.
to extract kinetic constants from the Kitz-Wilson analysis:42
incubated for 30 addition minutes. Each sample was applied to a
C18 ZipTip (Millipore) column, and eluted with 1:1 CH3CN/H2O
containing 0.05% TFA. The eluent was analyzed by MALDI-TOF
mass spectrometry in R-cyano-4-hydroxycinnamic acid.
Antibodies. Antibodies against mono-, di, and trimethylated
H3K4 were obtained from Abcam (Cambridge, MA).
kobs ) (kinact*[I])/(Ki(inact) + [I])
(2)
Ki(inact) was extrapolated to zero substrate by:
app
i(inact)
K
) Ki(inact)*(1 + S/Km)
(3)
(4)
Cell Culture, Transient Transfection, and Reporter
Analyses. Monolayer cultures of clone -23 cells derived from GH3
cells and containing a luciferase reporter construct under the control
of the TSHR subunit promoter (-840 to +1),38 were grown in
DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented
with 10% heat-inactivated fetal calf serum (Biofluids, Rockville,
MD) and maintained in 5% CO2 atmosphere at 37 °C. Cells were
transfected with 300 ng DNA/well in 12-well plates with Lipo-
fectamine 2000 (Invitrogen) according to the manufacturer’s
instructions. Cell culture media were changed 6 h after transfection
to antibiotic-free DMEM plus 10% charcoal-dextran-treated fetal
bovine serum. T3 was added to the cells 48 h later, and cells were
harvested, lysed and assayed for reporter gene activity the next day
using dual luciferase assay reagents according to the manufacturer’s
instructions (Promega Corp., Madison, WI).
Chromatin Immunoprecipitation (ChIP) Assay. ChIP assay
was performed as previously described.38 R-23 cells were grown
to 90% confluence in phenol red-free DMEM supplemented with
10% charcoal DEXTRAN-stripped fetal bovine serum (FBS) for 3
days. After addition of 10-6 M T3 and/or 10 µM phenelzine for
one hour, ChIP assays were performed according to manufacturer’s
protocol (Upstate Biotechnology) with some minor modifications.
After treatment with T3, chromatin was cross-linked with 1%
formaldehyde in PBS, and nuclei extracted. Chromatin was soni-
cated to generate 500- to 1000-bp DNA fragments, and the
supernatant containing precleared chromatin was incubated at 0 °C
overnight with different antibodies or rabbit IgG control. After
reverse cross-linking by heating the samples at 65 °C overnight,
and treating with Proteinase K, DNA was purified with a QIAGEN
PCR Purification Kit (QIAGEN, Chatsworth, CA) according to the
manufacturer’s instructions. Quantitative real-time PCR of ChIP
products was performed using primers that amplified the promoter
region of TSH in pGL2/TSH (upstream primer: 5′-CAGGATGT-
TATGTGTATGGCTC- 3′ and downstream primer: 5′-CTTTAT-
GTTTTTGGCGTCTTC-3′). We employed the SYBR green system
(Applied Biosystems) using an Applied Biosystems 7300 sequence
detector. Relative values (mean ( SD) normalized to input levels
were compared with those obtained with control IgG.
The t1/2 for inactivation at saturation was obtained by:
t1/2 ) (ln 2)/kinact
Compounds 7 and 8 did not display time-dependent inhibition.
Initial velocities at increasing concentrations of 7 and8 were
obtained by linear regression to reaction progress curves. These
velocities were used to determine the Ki for 7 and8 by Dixon
analysis assuming competitive inhibition. The Ki was extrapolated
to zero substrate by:
Kai pp ) Ki*(1 + S/Km)
(5)
Adduct Analysis by Absorbance Spectroscopy. GST-LSD1
(4.25 µM) was incubated with potential inactivators (30 µM), or
no inhibitor in 50 mM HEPES (pH 7.5) at 25 °C. After 1 h, the
samples were clarified by centrifugation at 14 000× g for 10 min
(25 °C) and the flavin absorbance spectra was recorded (350-550
nm).
MALDI-TOF Mass Spectrometric Analysis of Peptide
Inactivators. Aliquots of the GST-LSD1 that was inactivated by
peptides 3, 4, and 18 in the absorbance spectroscopic analysis above
were used for MALDI-TOF mass spectrometric analysis. The
sample was applied to a C18 ZipTip (Millipore) column, and eluted
with 75:25 CH3CN:H2O containing 0.05% TFA. The eluent was
analyzed by MALDI-TOF mass spectrometry in R-cyano-4-
hydroxycinnamic acid.
Preincubation Assay with Phenelzine. GST-LSD1 (10 µM) was
incubated with phenelzine (250 µM), or no inhibitor, in 50 mM
HEPES (pH 7.5) for 30 min at 25 °C. The inhibition study described
above was initiated by the addition of 3 µL of preincubated LSD1
to reaction mixtures consisting of substrate and cosubstrates for a
final phenelzine concentration of 5 µM in 150 µL. Two control
assays were performed, the first lacking phenelzine and the second
consisting of phenelzine present in the reaction mixture for 5 µM
final.
MALDI-TOF Preincubation Analysis with Phenelzine. GST-
LSD1 (185 nM) was incubated with phenelzine (500 µM), or no
inhibitor, in 50 mM HEPES (pH 7.5) at 25 °C. After 30 min
dimethyl-Lys-4 H3-21 substrate (10 µM final) was added and
Acknowledgment. We thank the NIH for financial support and
L. Szewczuk for helpful suggestions.
(42) Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245–3249.
JA909996P
9
3176 J. AM. CHEM. SOC. VOL. 132, NO. 9, 2010