Structure-based screening and optimization of cytisine derivatives as inhibitors of the menin-MLL interaction

Article abstract of DOI:10.1039/c6cc01079b

The natural product-like compound 1 was identified as a direct inhibitor of the menin-MLL interaction by in silico screening. Structure-based optimization furnished analogue 1a, which showed significantly higher potency than both the lead structure 1 and the reference compound MI-2.

Full text of DOI:10.1039/c6cc01079b

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Structure-based screening and optimization
of cytisine derivatives as inhibitors of the
Cite this:DOI: 10.1039/c6cc01079b
menin–MLL interaction†
Received 2nd February 2016,
Accepted 10th March 2016
Hai-Jing Zhong,a Bo Ra Lee,c Joshua William Boyle,c Wanhe Wang,b
Dik-Lung Ma,*b Philip Wai Hong Chan*cd and Chung-Hang Leung*a
DOI: 10.1039/c6cc01079b
www.rsc.org/chemcomm
The natural product-like compound 1 was identified as a direct reported, and some have been investigated for the treatment of
inhibitor of the menin–MLL interaction by in silico screening. leukemia.9,21,22 In particular, MI-503 and MI-463 show sub-
Structure-based optimization furnished analogue 1a, which showed stantial survival benefit in mouse models of MLL leukemia.23
significantly higher potency than both the lead structure 1 and the
reference compound MI-2.
In the literature, menin–MLL interaction inhibitors have
been identified by a fluorescence polarization (FP) assay with
the MBM1 peptide labeled with fluorescein. MI-2, the first-in-
The mixed-lineage leukemia proteins (MLL) are histone methyl- class inhibitor of the menin–MBM1 interaction, showed an IC50
transferases that act as epigenetic transcriptional regulators.1–3 value of 3.5 mM in the FP assay. X-ray crystallography analysis
H3K4me3 is methylated by the MLL SET domain, resulting in indicated that MI-2 is situated in the central cavity on menin in
the activation of genes, such as the HOX genes.4,5 Significantly, the MBM1 pocket, mimicking the critical interactions of
the menin–MLL complex was found to be substantially activated in MBM1.11,22
human hepatocellular carcinoma specimens, where it promotes
Virtual screening has become an essential tool in assisting
hepatocellular carcinoma development in an H3K4me3-dependent drug lead discovery and optimization in recent years. In this
manner.6,7 Loss of menin-binding ability abolishes the tumori- study, we sought to apply high-throughput, ligand-docking
genic potential of aaMLL fusion constructs both in vitro and based virtual screening methods to identify small agents targeting
in vivo.8 The menin–MLL interaction therefore represents an menin–MLL binding from a natural product/natural product-like
attractive therapeutic target for the development of novel drugs chemical database. The high-resolution crystal structure of
for cancers with MLL rearrangements, such as liver carcinomas.9,10 human menin in complex with MI-2 (PDB: 4GQ3)12 was utilized
Menin interacts with two fragments of MLL, menin-binding to generated a molecular model in this study. Over 90 000
motif (MBM) 1 (MBM1) and MBM2. Menin binds to MBM1 with natural product/natural product-like structures (AnalytiCon
over 26-times higher affinity compared to MBM2, which are both Discovery) were screened according to the internal coordinate
located within the intrinsically unstructured 43-amino acid fragment mechanics (ICM) method [ICM-Pro 3.6-1d program (Molsoft,
at the N-terminus of MLL.11 MBM1 and MBM2 simultaneously and San Diego, CA, USA)].24
competitively bind to menin with negative cooperativity.12,13 The
bivalent menin–MLL interaction has represented a challenge or the results were tested in a bimolecular fluorescence complementa-
discovery of small molecule menin–MLL inhibitors.14
tion (BiFC) assay to explore their effect on the menin–MLL
The 15 highest-scoring compounds from the virtual screening
Natural products offer a wide variety of chemical skeletons interaction (Fig. S1, ESI†).25 HepG2 cells were co-transfected with
with bioactivity and low toxicity.15–20 To date, several small mole- MLL-VC210 and VN210-menin to allow for the visualization of the
cule inhibitors targeting the menin–MLL interaction have been menin–MLL complex in cellulo. From this assay, compound 1
emerged as the top candidate for inhibiting menin–MLL inter-
action. (Fig. S2 and S3, ESI†).
a State Key Laboratory of Quality Research in Chinese Medicine,
Institute of Chinese Medical Sciences, University of Macau, Macao, China.
E-mail: duncanleung@umac.mo
We performed molecular modeling in order to further
elucidate the binding mode of 1 with menin. The lowest-
scoring binding pose of compound 1 in the MLL binding
pocket of menin is depicted in Fig. 1a. Intriguingly, a hydrogen
bond is predicted to be formed between the pyridone oxygen
atom of 1 with the side chain OH group of Tyr276. This
b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong,
Hong Kong, China. E-mail: edmondma@hkbu.edu.hk
c School of Chemistry, Monash University, Clayton, Victoria 3800, Australia.
E-mail: phil.chan@monash.edu
d Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc01079b H-bonding interaction with Tyr276 is also observed in the X-ray
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Fig. 2 BiFC assay comparing activities of compounds 1, 1a–1g and MI-2
(10 mM) for disruption of the menin–MBM1 interaction in HepG2 cells.
HepG2 cells were transiently transfected with MLL-VC210 and VN210-
menin plasmids. Comparative nuclei staining with DAPI is shown.
Fig. 1 (a) Low-energy binding conformations of compound 1 (left) and 1a
(right) bound to menin interface as generated by virtual ligand docking. (b)
Chemical structures of analogues of compound 1 (1a–1g).
crystal structure of MI-2 with menin, in which the N1 nitrogen Thus, we were interested to investigate whether structural
atom of the thienopyrimidine ring acts as a H-bond acceptor. analogues of compound 1 could form more effective interactions
Moreover, a high degree of shape complementarity is observed with the MLL binding pocket compared to 1 itself, hence leading
between compound 1 and the binding pocket of menin, suggesting to the discovery of more potent menin–MLL inhibitors. The
that this protein–ligand interaction could also be stabilized by commercially available (À)-cytisine (1b) along with six other
significant hydrophobic interactions, as was the case for MI-2.22
analogues (1a, 1c–g) (Fig. 1b) containing varying side-chains
Interestingly, compounds 1, 4 and 11 all possess the (À)-cyti- were designed and synthesized and were screened for their
sine (1b) moiety, and they all showed some degree of inhibition menin–MLL inhibitory activities. Interestingly, (À)-cytisine (1b)
of the menin–MLL interaction in the BiFC assay (Fig. S2 and S3, and all its derivatives 1a, 1c–1g showed some degree of inhibition
ESI†). This suggests that the (À)-cytisine motif may contribute of the menin–MLL interaction in the cell-based environment
to the high binding affinity of these analogues to menin. (Fig. 2 and Fig. S6, ESI†). This result confirms that the cytisine
Cytisine alkaloids have also been reported as catechol-O- alkaloid scaffold could be an important factor in determining
methyltransferase inhibitors.26 However, the observation that inhibitory activity against the menin–MLL interaction. Compound
the fluorine-containing 4 and the chlorine-containing 11 are 1a, bearing the 5-phenylpentan-2-ol side-chain, was found to
less potent than 1 may indicate that the presence of halogen completely block the menin–MLL interaction at a concentration
atoms are unfavourable for menin–MLL inhibitory activity. of 10 mM.
Compound 1 itself is not found in nature and to our knowl-
Based on the BiFC results for the analogues of compound 1,
edge, its biological properties have not been previously preliminary structure–activity relationships can be deduced.
reported. Besides these three cytisine-containing compounds, The most potent compound 1a is identical to 1 except that it
the bisphenol 5 and the coumarin 10 also showed moderate has a methylene group (CH2) in place of a sulfur atom in the
menin–MLL inhibitory activity in the BiFC assay, however these alkyl linker chain. This indicates that the presence of a sulfur
were not further pursued due to the promising potency of atom in the linker region is deleterious for menin–MLL inhibition,
compound 1.
as is the insertion of nitrogen (as in 1c) or oxygen (as in 1d) atoms
A FP assay was conducted to verify the potency of compound in the linker chain. Additionally, replacement of the OH group in
1 against the menin–MLL interaction. In this assay, a FITC- 1a with OPNB (1e), OMe (1f) or OBn (1g) all detracted from
labeled MLL-derived peptide encoding the high-affinity MBM1 biological potency. Interestingly, the cytisine motif (1b) itself was
fragment is reacted with menin that has been pre-incubated with highly active, though not as potent as 1a or 1, confirming that this
the compounds. Dose–response analysis showed that compound 1 structural motif is the driving force for menin–MLL inhibitory
disrupted the interaction of menin–MLL with an IC50 value of activity.
0.04 mM (Fig. S4, ESI†). In cytotoxicity evaluation, compound 1
exhibited potent antiproliferative activity against HepG2 hepato- binding pocket of menin is depicted in Fig. 1b. Interestingly,
cellular carcinoma cells (IC50 = 10 mM) (Fig. S5, ESI†). the binding pose of 1a is quite different to that of the original
The lowest-scoring binding pose of compound 1a in the MLL
In the docking diagram of compound 1 with menin (Fig. 1a), lead structure 1. Notably, the cytisine ring system of 1a is
we observed that the MLL binding site was relatively large and predicted to be rotated more deeply into the MLL binding
spacious, as is the case for most protein–protein interactions. pocket, leading to two distinct consequences. The first is that
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activity of 1a towards HepG2 cells was further examined using the
colony formation assay, where it showed an IC50 value of (IC50
=
0.6 mM) (Fig. S9, ESI†). We consider that the cytotoxicity afforded
by 1a could be attributed, at least in part, to the inhibition of the
menin–MLL interaction in cellulo.
In conclusion, in silico screening identified a cytisine alkaloid
1 as an inhibitor of the menin–MLL interaction. Subsequent
structure-based optimization afforded the significantly more
potent analogue 1a. 1a inhibited the interaction between menin
and MLL as revealed by multiple biochemical assays, including
BiFC, FP and co-IP assays. Additionally, 1a inhibited H3K4me3
and p27 expression in heptacellular carcinoma cells. Compound
1a also exhibited potent anti-proliferative activity in heptacellular
carcinoma cells, possibly through inhibition of the menin–MLL
interaction. To our knowledge, 1a is the second compound class
that has been reported to inhibit the menin–MLL interaction. We
envision that 1a may serve as a useful structural class for the
Fig. 3 (a) Compound 1a suppressed menin–MLL interaction in HepG2
cells as revealed by co-IP. (b) Compound 1a suppressed H3K4me3 and p27
levels in HepG2 cells.
the pyridone carbonyl group of 1a is predicted to be pushed generation of more effective therapeutic agents against cancers
away from Tyr276, abolishing the H-bonding interaction that is such as heptacellular carcinoma.
found between 1 and this residue. The second is that the
This work is supported by Hong Kong Baptist University
pendant phenyl ring and alkyl chain of 1a is nestled further (FRG2/14-15/004 and FRG2/15-16/002), the Health and Medical
towards the a-helix on the right side of the docking site. Given Research Fund (HMRF/14130522), the Research Grants Council
that 1a showed a greater inhibitory potency than compound 1, (HKBU/201811, HKBU/204612 and HKBU/201913), the French
it could be that this alternate binding mode is more conductive National Research Agency/Research Grants Council Joint
for menin–MLL inhibition.
Research Scheme (A-HKBU201/12), Inter-institutional Colla-
To further validate the inhibitory potency of 1a, the compound borative Research Scheme (RC-ICRS/15-16/02A), National Natural
was tested in a dose–response experiment using the FP assay. 1a Science Foundation of China (21575121), Guangdong Province
inhibited the menin–MLL interaction with an IC50 value of 1 nM Natural Science Foundation (2015A030313816), Hong Kong
(Fig. S7, ESI†).
Baptist University Century Club Sponsorship Scheme 2015, Inter-
We next performed a co-immunoprecipitation (co-IP) assay disciplinary Research Matching Scheme (RC-IRMS/14-15/06), the
using HepG2 cells co-expressing menin and MLL to investigate Science and Technology Development Fund, Macao SAR (103/
whether 1a could inhibit menin–MLL binding in cells. In the 2012/A3), the University of Macau (MYRG091(Y3-L2)-ICMS12-
absence of 1a, menin co-immunoprecipitated with MLL LCH, MYRG2015-00137-ICMS-QRCM, MRG023/LCH/2013/ICMS
(Fig. 3a). A dose-dependent decrease in the level of menin and MRG044/LCH/2015/ICMS) and a Discovery Project Grant
was observed upon treatment of HepG2 cells with 1a, thus (DP160101682) from the Australian Research Council.
suggesting that 1a was able to inhibit the binding between
menin and MLL in cells.
Given by the promising activity of 1a at antagonizing menin–
MLL binding in vitro, the compound was further examined for
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