A R T I C L E S
Binda et al.
expressed in Escherichia coli and purified as described.6 Human
recombinant LSD1/CoREST were expressed in E. coli as separate
proteins and co-purified following previously reported procedures.20
Enzymatic activities and inhibition assays with both demethylases
were carried out at pH 7.5-8.0 using methylated H3 peptides.6,20
Crystals of LSD1/CoREST inhibitor complexes were obtained by
cocrystallization under conditions identical to those previously
reported for other structural studies on these proteins.14,20 Diffrac-
tion data were measured at the European Synchrotron Radiation
Facility and the Swiss Light Source. Data processing and structure
refinement were carried out using standard procedures.20,21 Crystal-
lographic and refinement statistics together with the PDB accession
codes are reported in Table S5. Figures were prepared with Pymol
(www.pymol.org).
NB4 cells were treated at different concentrations of 14e (Table
1). Whole cell lysates were subjected to SDS-PAGE, and then
immunoblotted using antibodies against different histone modifica-
tions (from Abcam: H3K4me2, H3K9me2, H3; antiacetylated H4,
T2522). Analysis of NB4 cells and murine APL blasts growth and
differentiation was performed as described previously.23,24
Data Deposition. Coordinates have been deposited with the
Protein Data Bank. Accession codes are 2XAF, 2XAG, 2XAH,
2XAJ, 2XAQ, 2XAS (Table S5).
epigenetic drugs as suggested by its overexpression in solid
tumors,8 its role in various differentiation processes,9 its
involvement in herpes virus infection,10 and its association to
histone deacetylase 1, a validated drug-target. LSD2, like LSD1,
displays a strict specificity for mono- and dimethylated Lys4
of H3. However, the biology of LSD2 is proposed to differ from
that of LSD1 since LSD2 does not bind CoREST and has not
been found so-far in any LSD1-containing protein complex.6,11,12
LSD1 and LSD2 share a similar catalytic domain (45%
sequence identity) that is structurally homologous with the amine
oxidases, a class of flavin-dependent enzymes that act on
biogenic amines.4,5 Among these proteins, human monoamine
oxidases (MAOs) A and B have been the subject of more than
50 years of research that has led to the development of a
multitude of inhibitors including antidepressive and antiparkin-
son drugs.13 Their similarity in the catalytic and structural
properties prompted the investigation of antiMAO drugs as
potential LSD1 inhibitors.14 It was found that tranylcypromine
(Table 1), a MAO inhibitor used as antidepressive drug, is able
to inhibit LSD1.15-18 On this basis, we sought to design
compounds that would be more selective for demethylases using
tranylcypromine as the lead scaffold. Here, we report the
synthesis of a series (more than 40) of new tranylcypromine
analogues and a biochemical and biological evaluation of their
inhibitory properties with human LSD1, mouse LSD2, human
MAO A, and human MAO B (see Supporting Information). The
results demonstrate that many of these compounds are effective
LSD1 and LSD2 inhibitors, and most importantly, the prototype
14e (Table 1) exhibits synergistic activities with antileukemia
drugs.
Results
Enantioselectivity of the Inhibition. Tranylcypromine is a
racemic mixture of (()-trans-2-phenylcyclopropyl-1-amine ·HCl
(tPCPA) that covalently inhibits the LSD and MAO enzymes.
The first question we addressed in our study was the difference,
if any, between the two tPCPA enantiomers with respect to their
inhibition activities. (+)- and (-)-tPCPA were synthesized and
their absolute configuration determined (see Supporting Infor-
mation). Biochemical analysis showed that the inversion of the
configuration has a marginal effect on inhibition of the two LSD
enzymes, whereas it is significant for inhibition of MAO B.25
In addition, the crystallographic analysis of LSD1/CoREST
inhibitor complexes highlighted a surprising feature: the two
enantiomers differ both in the binding orientation and nature
of the covalent adduct with the flavin (Figure 1-3; Scheme 1).
This feature was confirmed using the para-brominated deriva-
tives whose electron-rich substituent provided enhanced clarity
of the electron density maps (Figure 1). (-)-tPCPA engages
its carbonyl carbon in a covalent bond with the flavin N5 atom
and positions its phenyl ring in the core of the substrate-binding
pocket, stacking above the flavin ring. In contrast, the phenyl
ring of the (+)-tPCPA binds in a lateral niche of the substrate-
binding pocket and points away from the flavin ring (Figure
1-3). Furthermore, the covalent bond with the flavin N5 atom
involves the phenyl-substituted carbon of the inhibitor rather
than the carbonyl carbon as observed for the (-)-enantiomer
(Scheme 1).
Materials and Methods
Syntheses of all tested compounds are described in the Supporting
Information. Human recombinant MAO A and MAO B were
expressed in Pichia pastoris and purified as published.19 Inhibition
assays and Ki values were measured using kynuramine (MAO A)
and benzylamine (MAO B) substrates at pH 7.5 according to
published procedures (Table 1).19 Mouse recombinant LSD2 was
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