Identification of novel inhibitors of histone acetyltransferase hMOF through high throughput screening

Article abstract of DOI:10.1016/j.ejmech.2018.08.026

The histone acetyltransferases (HATs) in mammals include GCN5 N-acetyltransferases, the MOZ, YBF2, SAS2, and TIP60 proteins, and the orphan HATs. The males absent on the first (MOF) is mainly related to acetylation of histone H4 Lys16 and has influence on downstream genes expression. However, the only inhibitor MG149 presented low activity against MOF. Besides, there was no high throughput screening platform on MOF, which limited the inhibitor discovery and functional study. In our study, we set up a high throughput screening platform based on amplified luminescent proximity homogeneous assay (ALPHA), which led us to a moderate inhibitor DC_M01. By chemical modification, we found DC_M01_7, which was the analog of DC_M01 with an IC₅₀ value of 6 μM. DC_M01_7 significantly inhibited HCT116 cells proliferation and could also inhibit histone 4 lysine 16 acetylation in HCT116 cells. To sum up, our work will probably assist the further development of more potent MOF inhibitors and the functional study of hMOF.

Full text of DOI:10.1016/j.ejmech.2018.08.026

Accepted Manuscript
Identification of novel inhibitors of histone acetyltransferase hMOF through high
throughput screening
Rukang Zhang, Jiang Wang, Liang Zhao, Shien Liu, Daohai Du, Hong Ding, Shijie
Chen, Liyan Yue, Yu-Chih Liu, Chenhua Zhang, Hong Liu, Cheng Luo
PII:
S0223-5234(18)30690-1
10.1016/j.ejmech.2018.08.026
EJMECH 10637
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2018
Revised Date: 25 July 2018
Accepted Date: 10 August 2018
Please cite this article as: R. Zhang, J. Wang, L. Zhao, S. Liu, D. Du, H. Ding, S. Chen, L. Yue, Y.-
C. Liu, C. Zhang, H. Liu, C. Luo, Identification of novel inhibitors of histone acetyltransferase hMOF
through high throughput screening, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.08.026.
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ACCEPTED MANUSCRIPT

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Identification of novel inhibitors of histone acetyltransferase hMOF
through high throughput screening
∥
Rukang Zhang,†, ,# Jiang Wang,†, # Liang Zhao,† Shien Liu,⊥ Daohai Du†,
Hong Ding†, Shijie Chen†, Liyan Yue†, Yu-Chih Liu,§ Chenhua Zhang,§ Hong
Liu,*, † and Cheng Luo*,†
†State Key Laboratory of Drug Research, CAS Key Laboratory of Receptor Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
China
∥University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
⊥WuXi AppTec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China
§Shanghai ChemPartner Co., Ltd., #5 Building, 998 Halei Road, Shanghai 201203, China.
ABSTRACT
The histone acetyltransferases (HATs) in mammals include GCN5 N-acetyltransferases, the MOZ,
YBF2, SAS2, and TIP60 proteins, and the orphan HATs. The males absent on the first (MOF) is
mainly related to acetylation of histone H4 Lys16 and has influence on downstream genes expression.
However, the only inhibitor MG149 presented low activity against MOF. Besides, there was no high
throughput screening platform on MOF, which limited the inhibitor discovery and functional study. In
our study, we set up a high throughput screening platform based on amplified luminescent proximity
homogeneous assay (ALPHA), which led us to a moderate inhibitor DC_M01. By chemical
modification, we found DC_M01_7, which was the analog of DC_M01 with an IC50 value of 6 µM.
DC_M01_7 significantly inhibited HCT116 cells proliferation and could also inhibit histone 4 lysine
16 acetylation in HCT116 cells. To sum up, our work will probably assist the further development of
more potent MOF inhibitors and the functional study of hMOF.
HIGHLIGHT
ò
ò
We identified a potent hMOF inhibitor with a new scaffold using high throughput screening.
The binding affinity of the hit compound DC_M01 was measured by SPR.

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ò
ò
DC_M01_7, which was obtained by chemical modification, could inhibit hMOF activity in a
substrate competitive mode.
DC_M01_7 could inhibit hMOF activity in HCT116 cells and regulate downstream genes.
KEYWORDS
High throughput screening; Epigenetics; Histone acetyltransferase; MOF; inhibitor;
INTRODUCTION
Histone acetylation which gives rise to DNA relaxation with a positive influence on transcription can
be regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) [1]. Among
these two kinds of enzymes, HATs can be divided into three groups, including the GNATs (Gcn5
N-acetyltransferases), the MOZ, YBF2, SAS2, and TIP60 proteins (MYSTs) and the orphan HATs [2].
MOF (males absent on the first), a member of the MYST family, was initially found to function by
acetylation of histone H4 Lys16 (H4K16ac) in dosage compensation whereby transcription of genes
on the single male X-chromosome must be increased two-fold relative to females who have two
X-chromosomes [3, 4]. HMOF, the homolog of the Drosophila dosage compensation proteins MOF,
forms human histone acetyltransferase complex (hMSL) which shows strong specificity for histone
H4 lysine 16 in chromatin in vitro and in vivo [5].
HMOF displays quite diverse roles in various nuclear processes and some have also been implicated
in carcinogenesis [6]. In comparison to normal tissues, hMOF is overexpressed in different kinds of
cancers, such as human oral tongue squamous cell carcinoma (OTSCC), non-small cell lung cancer
(NSCLC), colon cancer, and thymic lymphoma [7, 8]. In OTSCC, hMOF enhanced OSTCC growth
by targeting EZH2 [9]. In lung cancer cells A549 and H1299, hMOF RNAi reduced the migration and
adhesion. In addition, genes involved in cell proliferation, adhesion and migration like SKP2, ETS1
and ITAG2 were down-regulated. SKP2 is a subunit of SKP1-CUL1-F-box ubiquitin ligase complex
that involved in regulation of G1 to S phase transition. The complex is also a positive regulator of
proliferation [10]. Further experiments proved that hMOF promoted S phase entry via SKP2 in H1299
cells [8]. Another study revealed that hMOF mediated acetylation increased Nrf2 and its downstream
genes and led to large tumor size, advanced disease stage and poor prognosis in NSCLC patients [11].

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In breast cancer, MOF acetylated oncogene AIB1 and enhanced its function in promoting breast
cancer cells [12]. Besides, in Hela cells, oncogene HOXA9, UCP2, KIAA0657, and HIP1 were
down-regulated when cells were transfected with hMOF-specific siRNAs. Moreover, MOF depletion
results in decreased cell numbers post-irradiation in SL-2 cells [13]. H4K16 acetylation had close
relevance with breast cancer and colon cancer [14]. Analogs of SFN could sensitize HCT116 cells via
modulate HAT/HDAC activities and associate DNA damage/repair signaling pathways [15]. Histone
acetylation could upregulate the expression of NBL2 that was associate in colorectal cancer cells, such
as HCT116 cells [16]. Another study showed that histone acetylation modulated the transcriptional
activities of several tumor suppressors and immune modulatory genes that related in colorectal cancer
cells [17]. Also, a recent study showed that deletion of MOF in a mouse model of MLL-AF9 driven
leukemogenesis reduced tumor burden and prolonged host survival [18]. According to these data, we
concluded hMOF inhibitor may find application in the treatment of several types of cancer such as
leukemia, colon cancer, NSCLC. Therefore, searching for selective inhibitor of hMOF not only can
facilitate researching hMOF function in related diseases but also may contribute to make promising
tools for the treatment of diseases mentioned above.
Parallel to functional studies on HATs, researchers have aimed at developing small molecule inhibitors
as chemical probes or potential therapeutic agents [19]. The current HAT inhibitors can be classified to
three classes: bi-substrate, natural products, and novel small molecule inhibitors from virtual
screening, high throughput screening or structure-based design [20]. The first bi-substrate inhibitor of
HATs, H3-CoA-20 inhibitor was synthetized by Ronen Marmorstein’s group at 2002 with an IC50
value of 300 nM on tGCN5 [21]. In addition to bi-substrate analogs, several natural products have
been reported as HAT inhibitors. Anacardic acid from cashewnut shell liquid and garcinol from
Garcinia indica fruit rind were isolated by Kundu’s group, which has inhibition on p300 and PCAF,
respectively [22, 23]. Curcumin, a polyphenolic compound from curcuma longa rhizome, was shown
to be a specific inhibitor of p300/CBP HAT activity with an IC50 value of 25 µM [24]. However,
unlike natural products, a limited number of novel small molecules have been described as HATi.
MB-3 has been discovered as a GCN5 inhibitor with an IC50 value of 100 µM [25]. Isothiazolones as

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inhibitors of PCAF and p300 showed antiproliferative properties against a panel of human colon and
ovarian tumor cell lines [26]. A series of garcinol analogues (the LTK compounds) has been reported
as p300-specific HAT inhibitors (IC50 values = 5-7 µM) but inactive for PCAF [27]. Quinoline
derivatives have been described as HATi [28-30]. However, all these HATis reported have low
selectivity and could not be made into drug due to their high toxicity and low-permeability.
As for hMOF inhibitor’s study, Dekker and Zheng et al. synthesized MG149 (Figure 1) as an inhibitor
of Tip60 and MOF which has an IC50 value of 74 ± 20 µM and 47 ± 14 µM [31]. However, MG149
has no experimental data on hMOF in cell or in vivo. The lack of hMOF inhibitor limits the function
study of hMOF and also restricts the treat of related diseases.
Amplified luminescent proximity homogeneous assay (ALPHA) donor beads produced approximately
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60000 O2 that can spread up to 200 nM to excite acceptor beads under 680 nM laser. ALPHA has
become one of the most important methods in HTS due to its sensitivity, quickness and convenience
[32-35]. In our study, we set up a high throughput screening (HTS) platform based on ALPHA and
found a small molecule inhibitor DC_M01 of hMOF with an IC50 value of 40 µM. The binding
affinity of DC_M01 with hMOF is 19 µM in vitro. Then we synthesized a series of compounds based
on DC_M01 and found the DC_M01_7, which inhibited hMOF in vitro with an IC50 value of 6 µM.
What’s more, it can also dose-dependent inhibit hMOF in-cell. Taken together, our study indicates that
DC_M01_7 and its analogs are new potential hMOF inhibitors, which exhibit the best inhibitory
activity towards hMOF among all the reported hMOFi. This can provide us with new structural clues
to develop more potent hMOF inhibitors and may help the function study of hMOF in future.
RESULTS AND DISCUSSION
High throughput screening based on ALPHA.
As is mentioned before, the inhibitors of hMOF not only are in small quantity, but also have weak
activity. Due to these reasons, we lack of small molecules for studying the function of hMOF and
related diseases. To solve the problem of low efficiency of small molecule discovering, we set up a
high throughput screening platform based on ALPHA (Figure S1). This is the first high throughput
screening platform of hMOF which can probably accelerate the discovery of small molecule inhibitors

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of hMOF even the function study of hMOF.
We screened our in-house small molecule library which contains more than 20000 compounds using
this high throughput screening platform. The top 208 hit compounds from the first round screening
were picked up and conducted the second round screening. After taking out the compounds with
symmetrical structure and the compounds not suitable for structural modification, the hit DC_M01
with novel scaffold and best activity was picked up for further validation (Table S1).
Radioactive acetylation Assay.
To further validate the hit compounds, we performed radioactive acetylation assay and measured the
hit compound previously mentioned. DC_M01 showed the activity with an IC50 value of 40 µM
(Figure 1). This result further proved that DC_M01, can inhibit hMOF activity in vitro. So we choose
the hit DC_M01 to conduct further experiments for validation. In addition, the IC50 value of MG149
was also measured in radioactive acetylation assay (Figure S2).
Surface Plasmon Resonance (SPR) Based Binding Assay.
To validate DC_M01 as a hMOF inhibitor, we used the SPR-based binding assay to measure the direct
interaction between hMOF and DC_M01. The interactions of DC_M01 was dose-dependent and the
equilibrium dissociation constant (Kd) is 19 µM (Figure 1). The Kd of DC_M01 was consistent with
the hMOF inhibition activity.
Chemical Modification.
To improve the inhibitory activity of human MOF (hMOF), a series of novel analogs
DC_M01_1-DC_M01_7 was designed with incorporation of various substituents on the
benzothiophene ring based on the structure of hit compound DC_M01. Then, DC_M01_8-
DC_M01_15 were subsequently achieved via introducing various substituents to the phenyl ring and
replacing the N,N-diethyl sulfonamide fragment with different sulfonamide while keeping the
benzothiophene scaffold (Figure 2). The general synthetic route to DC_M01-DC_M01_12 is described
in Scheme 1. Condensation of the commercially available substituted 2-methylbenzothiazole
derivatives 2 and benzaldehyde 3 afforded the corresponding (E)-2-styryl benzothiazole 4.
Subsequently, compound 4 was reacted with excess chlorosulfonic acid to generate sulfonyl chlorides

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5. Finally, the target molecules DC_M01-DC_M01_12 were obtained via condensation of sulfonyl
chlorides 5 with different amines 6. Compounds DC_M01_13- DC_M01_15 were synthesized
according to Scheme 2. The intermidiate 8 was first afforded via condensation of sulfonyl chlorides 7
with diethylamine. Condensation of the commercially available 2-methylbenzo[d]thiazole 9 with
formaldehyde gave the 2-vinylbenzo[d]thiazole 10. Target compounds DC_M01_13-DC_M01_15
were generated by the coupling reaction of intermidiate 8 with 2-vinylbenzo[d]thiazole 10.
SAR Analysis.
In order to assess the inhibitory activity of DC_M01_1-DC_M01_15 against hMOF, biochemical
assays were performed as described in the experiment section. Compound potency against the hMOF
was interrogated using MG149 as the positive control. The results were presented in Table 1. Firstly,
hMOF inhibitory activity of the synthesized compounds that halogen was installed at the 5-position on
the benzothiophene ring was tested. To our delight, compounds DC_M01, DC_M01_01, and
-DC_M01_2 exhibited 53%, 64, and 57% hMOF inhibition at 50 µM, which displayed moderate
inhibitory activities against hMOF with IC50 values lower than 50. Introducing methyl (DC_M01_3)
and methoxyl group (DC_M01_4) at the 5-position on the benzothiophene decreased the inhibitory
activity. Compared with compound DC_M01_2, introduction of bromine (DC_M01_5) at the
6-position decreased the hMOF inhibitory activity. The mono-bromo substituted compounds
(DC_M01_2 and DC_M01_5) demonstrated the regiochemical preference of the benzothiphene
5-position > 6-position. Compounds DC_M01_6 and DC_M01_7 exhibited good inhibitory activity
with IC50 values of 7.7 µM and 6 µM, which is better than the positive control MG149 (IC50 = 15 µM).
Then, the SAR at the sulfamide moiety was investigated. Replacement of N,N-diethyl sulfonamide
moiety in DC_M01_7 with various sulfonamide afforded compounds DC_M01_8-DC_M01_12,
which decreased the hMOF inhibitory activity. Finally, introduction of fluorine, chlorine, and methyl
group at the 2-position of phenyl ring successively, compounds DC_M01_13-DC_M01_15 also
decreased the hMOF inhibitory activity.
Radioactive competitive assay.
In order to identify the binding pocket of DC_M01_7, we conducted the radioactive competitive assay

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to measure whether the DC_M01_7 could bind to the pocket of acetyl coenzyme A or the pocket of
histone 4. The increase concentration of acetyl coenzyme A did not affect the inhibition of DC_M01_7
(Figure 3A). However, the increase concentration of histone 4 peptide decreased the activity of
DC_M01_7. DC_M01_7 showed no inhibition on hMOF when the concentration of peptide was 10
µM (Figure 3B). This result illustrated that the DC_M01_7 inhibits hMOF by competitively
occupying the binding pocket of histone 4.
Docking Studies
Based on the radioactive competitive assay, the binding pocket of compound DC_M01_07 was
determined. Compounds DC_M01, DC_M01_6, DC_M01_7, and DC_M01_15 were selected to dock
into the hMOF to investigate the binding modes and the interactions (Figure 4). It was observed that
compound DC_M01 occupied the active binding pocket of histone 4 (Figure 4A). The N,N-diethyl
sulfonamide moiety was forming hydrogen bonds with residues Ile144 and Ser181. Docking results
showed that 4-fluorobenzothiazole formed extensive hydrophobic interactions with Aly101 (Fig. 4A).
Compared with compound DC_M01, compounds DC_M01_6 and DC_M01_7 displayed the same
binding mode with the active site of hMOF (Figures 4B and 4C). However, introduction of methyl
group at the 2-position of phenyl ring, compound DC_M01_15 didn’t form the hydrogen bond with
Ile144, which is correlated with the decreased inhibitory activity (Figure 4D).
Colony formation assay.
According to previous study, hMOF in is overexpressed HCT116 cells.7 To investigate whether
compound can inhibit the proliferation of cancer cells, so we chose HCT116 cells to conduct the
colony formation assay. We used DC_M01_7 to treat HCT116 cells with different concentrations for
14 days and found that DC_M01_7 can dose-dependently inhibit the colony formation of HCT116
cells (Figure 5). However, the compound mg149 inhibits the colony formation only under
concentration of 100 µM (Figure S4). This result indicated that DC_M01_7 led to cell death in
HCT116 cells dose-dependently.
HMOF in cell inhibition Assay.
To confirm the DC_M01_7 can inhibit hMOF in cells, we conducted Western blot assays. After

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treating HCT116 cells for 24 hours with SAHA and DC_M01_7, the histone 4 lysine 16 acetylation
was measured using western blot assays. We observed a dose-dependently decrease of H4 lysine 16
acetylation (Figure 6A). This result demonstrated that DC_M01_7 could inhibit the hMOF activity
because in cellular context hMOF is the only known enzyme to modify this site.
To further explore the antiproliferation mechanism of DC_M01_7, we examined whether the
compound could block the oncogenes’ expression in HCT116 cells. According to researches
mentioned previously, hMOF knockdown leads to down-regulated of SKP2 and UCP2 that were
regarded as oncogenes in cancer cells [7, 8, 36]. So we measured the compounds’ effect on these
genes’ mRNA levels by using quantitative real-time PCR analysis. After treating HCT116 cells with
DC_M01_7 for 3days, the mRNA levels of SKP2 and UCP2 were dose-dependent down-regulated.
Meanwhile, the transcription level of IFI16 was up-regulated because IFI16 was reported to inhibit
cell growth and mediate apoptosis by p53 pathway [37, 38] (Figure 6B).
CONCLUSION
Histone acetylation catalyzed by HATs plays important roles in crucial cellular processes, such as
embryonic development, DNA damage repairing, and it is also involved in cancer development. Most
HAT inhibitors reported to date have low bioavailability. In our study, using the high throughput
screening approach based on ALPHA technology, we identified DC_M01 as a novel inhibitor of
hMOF with an IC50 value of 40 µM. To validate the hit DC_M01, we then performed radioactive
hMOF inhibition assay. To further confirm the binding mode of DC_M01 with hMOF, we conducted
SPR experiments and the Kd of DC_M01 was determined with 19 µM. After chemical modification,
we synthesized a series of DC_M01’s derivatives of which the DC_M01_7 has the IC50 value of 6 µM.
The DC_M01_7 also can led to cell death in HCT116 cells dose-dependently. To identify whether
DC_M01_7 can work in cancer cells on hMOF, we detected the western blot assay and observed a
dose-dependently decrease of H4 lysine 16 acetylation in HCT116 cells treated with DC_M01_7 in
different concentrations. Also, the qPCR assay illustrated that DC_M01_7 could regulate the mRNA
level of SKP2, UCP2, and IFI16.
Taken together, we found DC_M01_7 and its analogs are new hMOF inhibitors of which DC_M01_7

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shows best activity with an IC50 value of 6 µM. These compounds provided us with new structural
clues to develop more potent hMOF inhibitors.
EXPERIMENTAL SECTION
Plasmid Construction, Protein Expression, and Purification.
The plasmid was given as a gift by Ronen Marmorstein. The catalytic domain of hMOF (173-458)
was cloned to pRSFBuetTEVa vector using BamH1 and Sal1 with an N-terminal hexahistidine (His6x)
tag. The fusion protein was expressed in Escherichia coli BL21 (DE3) cells cultured at 37 and was
inducted at 16 in the presence of 0.4 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) when the
OD600 reached 0.6. Cells were harvested 16 h and lysed by sonication in lysis buffer containing 20
mM HEPES (pH 7.4), 500 mM NaCl, 10 mM imidazole, 0.1% β-mercaptoethanol. The protein was
first loaded on a HisTrap FF column (GE Healthcare), then washed using washing buffer (20 mM
HEPES (pH 7.4), 5000 mM NaCl, 60 mM imidazole, 0.1% β-mercaptoethanol) and the fusion protein
was eluted with elusion buffer (20 mM HEPES (pH 7.4), 500 mM NaCl , 350 mM imidazole , 0.1%
β-mercaptoethanol). The eluted protein was purified further by Superdex75 column (GE Healthcare)
in the buffer consisting 20 mM HEPES (pH 7.4), 200 mM NaCl, and 1 mM DTT. The purified protein
was stored at -80 °C in buffer (20 mM HEPES (pH 7.4), 200 mM NaCl, 1 mM DTT, 5% glycerol).
Radioactive hMOF Inhibition Assay.
The radioactive hMOF acetylation inhibition assay was performed in 20 µL reaction system
containing Acetyl Coenzyme A, [Acetyl-3H] - ([3H] Ac-CoA, 8.6 Ci/mmol, PerkinElmer),
biotinylated H4 derived peptide (synthesized by GL China) and KAT8 (Produced by SIMM) in
modified Tris Buffer, pH 7.5. The protein was pre-incubated with a range of compound concentrations
for 15 min at room temperature before adding biotinylated peptide and [3H] Ac-CoA. After 240 min
of incubation at room temperature, the reaction was stopped and was transferred to a 384-well
streptavidin-coated Flashplate (PerkinElmer) and was incubated for another DC_M01_7 at room
temperature. The Flashplate was washed with dH2O + 0.1% Tween-20 for three times and the
radioactivity was determined by liquid scintillation counting (MicroBeta, PerkinElmer). IC50 values
were derived by fitting the data for the inhibition percentage to a dose-response curve by nonlinear

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regression in GraphPad Prism 5.0.
Surface Plasmon Resonance (SPR) Based Binding Assays.
The SPR binding assays were performed on a Biacore T200 instrument (GE Healthcare) at 25 °C as
described. HMOF protein was covalently immobilized on a CM5 chip using a standard
amine-coupling procedure in 10 mM sodium acetate (pH 4.0). The chip was equilibrated with HBS
buffer (20 mM HEPES (pH 7.4), 200 mM NaCl, 0.5% (v/v) DMSO). For kinetic measurement,
compounds were diluted at concentrations ranging from 5 µM to 100 µM in HBS buffer. The diluted
compounds were injected over the chip for 120 s at a flow rate of 30 µL/min and then dissociate for
300 s. The state model of the T200 evaluation software was used to analyze the resulting data to
calculate the Kd of the compounds.
General Information.
The reagents (chemicals) were purchased from commercial sources, and used without further
purification. Analytical thin layer chromatography (TLC) was HSGF 254 (0.15-0.2 mm thickness). All
1
products were characterized by their NMR and MS spectra. H and 13C NMR spectra were recorded
on 400 MHz, 500 MHz, and 600 MHz instrument. Chemical shifts were reported in parts per million
(ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are described as singlet (s),
doublet (d), triplet (t), quartet (q), doublet doublet (dd), multiplet (m). Low- and high-resolution mass
spectra (LRMS and HRMS) were measured on spectrometer. HPLC analysis of all final biological
testing compounds was carried out on an Agilent 1100 Series HPLC with an Agilent Extend-C18
column (150×4.6 mm, 5 µm). All final compounds achieved a minimum of 95% purity (Table S3).
General procedure A: Synthesis of compounds DC_M01–DC_M01_12.
(E)-5-fluoro-2-styrylbenzo[d]thiazole (4a)
To a mixture of 5-fluoro-2-methylbenzo[d]thiazole 2a (224 mg, 1.34 mmol) and benzaldehyde 3 (142
mg 1.34 mmol) was added sodium amide (78 mg, 2 mmol) dissolved in dry DMF (5 mL), and the
mixture was refluxed for 1 h. The reaction was cooled to ambient temperature, then the reaction
solution was poured into ice-water (20 mL) to give crude product as yellow solid. The solid was
subsequently recrystallized from ethanol to give (E)-5-fluoro-2-styrylbenzo[d]thiazole 4a.

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Yellow solid (232 mg, yield 68%): m.p. 121-125 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.11-8.06 (m,
1H), 7.97 (d, J = 8.1 Hz, 1H), 7.78-7.74 (m, 2H), 7.70-7.58 (m, 2H), 7.54-7.48 (m, 1H), 7.45-7.40 (m,
2H). LRMS m/z: 256 [M + H]+.
(E)-N,N-diethyl-4-(2-(5-fluorobenzo[d]thiazol-2-yl)vinyl)benzenesulfonamide (DC_M01).
(E)-5-fluoro-2-styrylbenzo[d]thiazole DC_M01 (250 mg, 0.98 mmol) was added to a solution of the
chlorosulfonic acid (3 mL) at 0°C for 5 minutes. The solution was stirred at room temperature for 8 h
and then the mixture was poured into ice bath. Solid was precipitated and immediately collected
without further purification. The yellow solid, that is sulfonyl chloride, was continuously reacted with
diethylamine (98 mL, 9.8 mmol) in acetone. After 8 h, the mixture was poured into dilute hydrochloric
acid and the resulting precipitate was filtered. The crude product was stirred in 1 M sodium hydroxide
solution, and filtered as well followed by washing. The residue was purified by flash column
chromatography (PE/EA = 10 : 1) to yield the desired product DC_M01 (268 mg, yield 70%).
General procedure B: Synthesis of compounds DC_M01_13–DC_M01_15.
4-bromo-N,N-diethyl-2-fluorobenzenesulfonamide (8n)
To a solution of 4-bromo-2-fluorobenzene-1-sulfonyl chloride 7n (100 mg, 0.37 mmol) in acetone (5
mL) was added diethylamine (270 mg, 3.7 mmol) dropwise. The mixture was stirred at ambient
temperature for 5 h, and 1 N HCl (10 mL) was added to quench the reaction. The mixture was
extracted with dichloromethane (20 mL) for three times, and the combined organic layer was washed
with saturated NaHCO3 solution and brine. The organic solution was subsequently dried over
anhydrous Na2SO4 and concentrated in vacuo. The resulting residue was purified by flash column
chromatography (PE/EA = 10 : 1) to give 8n.
1
Pale yellow solid (103 mg, yield 90%): m.p. 68-71 °C, H NMR (400 MHz, CDCl3) δ 7.71-7.58 (m,
4H), 3.23 (q, J = 7.1 Hz, 4H), 1.13 (t, J = 7.1 Hz, 6H). LRMS m/z: 310 [M + H]+.
2-vinylbenzo[d]thiazole (10)

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Formaldehyde (270 mg, 8.72 mmol), triethylamine (33.5 mg, 0.34 mmol), and diethylamine
hydrochloride (790 mg, 8.72 mmol) were added to a solution of 2-methylbenzothiazole 9 (1.0 g, 6.71
mmol) in 1,4-dioxane (20 mL), and the mixture was stirred under 100 oC for 4 h. Water was added and
the mixture was extracted with dichloromethane (30 mL) for three times. The combined organic layer
was washed with brine, dried over anhydrous Na2SO4 and concentrated under vacumn. The crude
residue was purified by flash column chromatography (PE/EA = 50 : 1) to give compound 10.
1
White solid (767 mg, yield 71%): m.p. 25-26 °C, H NMR (400 MHz, CDCl3): δ 8.01-7.84 (m, 2H),
7.40-7.31 (m, 2H), 6.93 (dd, J = 16.8, 10.1 Hz, 1H), 5.93 (d, J = 13.8, Hz, 1H), 5.49 (dd, J = 16.7,
13.8 Hz, 1H). LRMS m/z: 161 [M + H]+.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N,N-diethyl-2-fluorobenzenesulfonamide (DC_M01_13)
To a solution of 4-bromo-N,N-diethyl-2-fluorobenzenesulfonamide 8n (100 mg, 0.32 mmol) in DMF
(5 mL) was added 2-vinylbenzothiazole 10 (61.8 mg, 0.38 mmol). After all the solid was dissolved,
palladium acetate (7.1 mg, 0.032 mmol), (±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (39.8 mg,
0.64 mmol), and Et3N (10 mg, 0.1 mmol) were added subsequently. The mixture was stirred at room
temperature for 8 h. Water (20 mL) was added into the solution, and the mixture was extracted with
dichloromethane (20 mL) for three times. The organic layer was separated, washed with brine, dried
over anhydrous Na2SO4. The crud residue was purified by flash column chromatography (PE/EA = 10 :
1) to give DC_M01_13 (75 mg, yield 60%).
(E)-N,N-diethyl-4-(2-(5-fluorobenzo[d]thiazol-2-yl)vinyl)benzenesulfonamide(DC_M01).
Synthesized by following general procedure A: Yellow solid (268 mg, yield 70%): m.p. 195-198 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.19 (dd, J = 8.9, 5.3 Hz, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.89-7.82
(m, 3H), 7.81-7.79 (m, 2H), 7.42-7.36 (m, 1H), 3.20 (q, J = 7.1 Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H). 13
C
NMR (125 MHz, DMSO-d6) δ 168.5, 161.4, (d, J = 243.6 Hz), 154.2, 140.0, 139.0, 135.8, 130.2,
128.4, 127.2, 124.3, 123.7, 114.2, 108.5, 41.8, 14.1. ESI-MS m/z: 391 [M + H]+. ESI-HRMS calcd for
C19H20FN2O2S2 [M + H]+ 391.0945, found 391.0949.

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(E)-4-(2-(5-chlorobenzo[d]thiazol-2-yl)vinyl)-N,N-diethylbenzenesulfonamide(DC_M01_1).
Synthesized by following general procedure A: Yellow solid (262 mg, yield 70%): m.p. 291-294 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 1.9 Hz, 1H), 8.11 (d, J = 8.5 Hz, 1H), 7.99 (d, J = 8.6
Hz, 2H), 7.84-7.80 (m, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.65-7.62 (m, 1H), 7.62-7.60 (m, 1H), 3.17 (q, J
= 7.1 Hz, 4H), 1.03 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 167.9, 154.6, 140.0, 139.0,
136.2, 133.5, 128.5, 127.2, 126.1, 125.0, 124.3, 124.1, 119.4, 41.8, 14.1. ESI-MS m/z: 407 [M + H]+.
ESI-HRMS calcd for C19H20ClN2O2S2 [M + H]+ 407.0649, found 407.0654.
(E)-4-(2-(5-bromobenzo[d]thiazol-2-yl)vinyl)-N,N-diethylbenzenesulfonamide(DC_M01_2).
Synthesized by following general procedure A: Yellow solid (236 mg, yield 66%): m.p. 175-178 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 2.0 Hz, 1H), 8.01 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.7
Hz, 1H), 7.84 (d, J = 8.2 Hz, 2H), 7.80 (s, 2H), 7.70 (dd, J = 8.7, 2.1 Hz, 1H), 3.20 (q, J = 7.1 Hz, 4H),
1.06 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 166.8, 152.4, 140.0, 139.1, 136.3, 136.0,
129.8, 128.4, 127.2, 124.9, 124.3, 124.2, 118.3, 41.8, 14.1. ESI-MS m/z: 451 [M + H]+. ESI-HRMS
calcd for C19H20BrN2O2S2 [M + H]+ 451.0144, found 451.0156.
(E)-N,N-diethyl-4-(2-(5-methylbenzo[d]thiazol-2-yl)vinyl)benzenesulfonamide (DC_M01_3).
Synthesized by following general procedure A: Yellow solid (278 mg, yield 72%): m.p. mp
1
256-270 °C. H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 8.3 Hz, 1H),
7.83-7.79 (m, 2H), 7.72 (d, J = 10.6 Hz, 2H), 7.57-7.63 (m, 1H), 7.37-7.32 (m, 1H), 3.17 (q, J = 7.1
Hz, 4H), 2.44 (s, 3H), 1.03 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 164.7, 151.6, 139.7,
139.2, 135.6, 134.9, 134.4, 128.2, 127.2, 126.1, 124.7, 122.3, 121.8, 41.8, 21.1, 14.1. ESI-MS m/z:
387 [M + H]+. ESI-HRMS calcd for C20H23N2O2S2 [M + H]+ 387.1195, found 387.1200.
(E)-N,N-diethyl-4-(2-(5-methoxybenzo[d]thiazol-2-yl)vinyl)benzenesulfonamide (DC_M01_4).
Synthesized by following general procedure A: Yellow solid (248 mg, yield 66%): m.p. 163-165 °C.

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1H NMR (400 MHz, DMSO-d6) δ 8.00 (dd, J = 8.6, 1.9 Hz, 3H), 7.83 (d, J = 8.3 Hz, 2H), 7.75 (d, J =
5.1 Hz, 2H), 7.55 (d, J = 2.5 Hz, 1H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H), 3.87 (s, 3H), 3.20 (q, J = 7.0 Hz,
4H), 1.06 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 166.8, 158.9, 154.8, 139.8, 139.2,
134.8, 128.3, 127.2, 126.1, 124.7, 122.6, 115.5, 105.4, 55.5, 41.8, 14.1. ESI-MS m/z: 403 [M + H]+.
ESI-HRMS calcd for C20H23N2O3S2 [M + H]+ 403.1145, found 403.1134.
(E)-4-(2-(6-bromobenzo[d]thiazol-2-yl)vinyl)-N,N-diethylbenzenesulfonamide (DC_M01_5).
Synthesized by following general procedure A: Yellow solid (232 mg, yield 65%): m.p. 167-168 °C.
1H NMR (500 MHz, DMSO-d6) δ 8.55-8.53 (m, 1H), 8.10 (d, J = 8.2 Hz, 2H), 8.04 (d, J = 8.7 Hz,
1H), 7.93 (d, J = 8.2 Hz, 2H), 7.88 (s, 2H), 7.81-7.77 (m, 1H), 3.30 (q, J = 7.1 Hz, 4H), 1.15 (t, J = 7.1
Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 167.9, 154.6, 140.0, 139.0, 136.2, 133.5, 128.5, 128.4,
127.2, 125.0, 124.2, 124.1, 119.4, 41.8, 14.1. ESI-MS m/z: 451 [M + H]+. ESI-HRMS calcd for
C19H20BrN2O2S2 [M + H]+ 451.0144, found 451.0142.
(E)-4-(2-(5,6-dimethylbenzo[d]thiazol-2-yl)vinyl)-N,N-diethylbenzenesulfonamide (DC_M01_6).
Synthesized by following general procedure A: Yellow solid (272 mg, yield 72%): m.p. 201-206 °C.
1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 8.3 Hz, 2H), 7.89-7.77 (m, 4H), 7.75-7.68 (m, 2H), 3.20
(q, J = 7.1 Hz, 4H), 2.37 (s, 6H), 1.06 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 164.5,
152.2, 139.7, 139.3, 135.6, 135.2, 134.6, 131.7, 128.2, 127.2, 124.9, 122.8, 121.8, 41.8, 19.7, 14.1.
ESI-MS m/z: 401 [M + H]+. ESI-HRMS calcd for C21H25N2O2S2 [M + H]+ 401.1352, found 401.1349.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N,N-diethylbenzenesulfonamide (DC_M01_7).
Synthesized by following general procedure A: Yellow solid (251 mg, yield 64%): m.p. 189-192 °C.
1H NMR (500 MHz, DMSO-d6) δ 8.15-8.12 (m, 1H), 8.04-7.99 (m, 3H), 7.85-7.81 (m, 2H), 7.79-7.76
(m, 2H), 7.57– 7.53 (m, 1H), 7.49-7.45 (m, 1H), 3.20 (q, J = 7.1 Hz, 4H), 1.06 (t, J = 7.1 Hz, 6H). 13C
NMR (125 MHz, DMSO-d6) δ 165.8, 153.4, 139.8, 139.2, 135.4, 134.3, 128.3, 127.2, 126.6, 125.7,
124.6, 122.7, 122.3, 41.8, 14.1. ESI-MS m/z: 373 [M + H]+. ESI-HRMS calcd for C19H21N2O2S2 [M +

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H]+ 373.1039, found 373.1041.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N,N-dimethylbenzenesulfonamide (DC_M01_8)
Synthesized by following general procedure A: Yellow solid (271 mg, yield 58%): m.p. 208-211 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 7.9 Hz, 1H), 8.08-8.00 (m, 3H), 7.86 -7.74 (m, 4H),
7.58-7.51 (m, 1H), 7.50 -7.45 (m, 1H), 2.64 (s, 6H). 13C NMR (125 MHz, DMSO-d6) δ 165.7, 153.4,
139.6, 135.3, 134.8, 134.3, 128.3, 128.0, 126.7, 125.8, 124.8, 122.8, 122.3, 37.6. ESI-MS m/z: 345 [M
+ H]+. ESI-HRMS calcd for C17H17N2O2S2 [M + H]+ 345.0726, found 345.0717.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N-phenylbenzenesulfonamide (DC_M01_9)
Synthesized by following general procedure A: Yellow solid (267 mg, yield 70%): m.p. 209-213 °C.
1H NMR (400 MHz, DMSO-d6) δ 10.33 (s, 1H), 8.12 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 8.1 Hz, 1H),
7.93 (d, J = 8.2 Hz, 2H), 7.80-7.65 (m, 4H), 7.53 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.24 (t, J
= 7.8 Hz, 2H), 7.14-7.09 (m, 2H), 7.03 (t, J = 7.4 Hz, 1H). 13C NMR (125 MHz, DMSO-d6) δ 165.7,
153.4, 139.5, 139.4, 137.6, 135.3, 134.2, 129.2, 128.2, 127.2, 126.6, 125.7, 124.7, 124.2, 122.8, 122.3,
120.2. ESI-MS m/z: 391 [M - H]-. ESI-HRMS calcd for C21H15N2O2S2 [M - H]- 391.0580, found
391.0588.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N-benzylbenzenesulfonamide (DC_M01_10)
Synthesized by following general procedure A: Yellow solid (263 mg, yield 70%): m.p. 225-229 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.27-8.22 (m, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.05-7.96 (m, 3H), 7.81
(dd, J = 23.1, 7.3 Hz, 4H), 7.59-7.53 (m, 1H), 7.51-7.45 (m, 1H), 7.33-7.20 (m, 5H), 4.03 (d, J = 6.2
Hz, 2H). 13C NMR (125 MHz, DMSO-d6) δ 165.8, 153.4, 140.8, 138.9, 137.6, 135.5, 134.2, 128.2,
128.2, 127.6, 127.1, 127.0, 126.7, 125.7, 124.4, 122.7, 122.3, 46.1. ESI-MS m/z: 405 [M - H]-.
ESI-HRMS calcd for C22H17N2O2S2 [M - H]- 405.0737, found 405.0740.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N-(4-methylbenzyl)benzenesulfonamide (DC_M01_11)

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Synthesized by following general procedure A: Yellow solid (195 mg, yield 53%): m.p. 199-202 °C,
1
HPLC: 99.8%. H NMR (400 MHz, DMSO-d6) δ 8.13 (d, J = 7.8 Hz, 2H), 8.02 (d, J = 8.1 Hz, 1H),
7.98-7.94 (m, 2H), 7.83-7.75 (m, 4H), 7.58-7.52 (m, 1H), 7.50 -7.44 (m, 1H), 7.14 (d, J = 8.5 Hz, 2H),
6.86-6.80 (m, 2H), 3.94 (s, 2H), 3.70 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 165.8, 158.4, 153.4,
140.9, 138.8, 135.5, 134.2, 129.3, 128.9, 128.1, 127.0, 126.6, 125.7, 124.4, 122.7, 122.3, 113.6, 55.0,
45.7. ESI-MS m/z: 419 [M - H]-. ESI-HRMS calcd for C23H19N2O2S2 [M - H]- 419.0899, found
419.0902.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N-(4-methoxyphenyl)benzenesulfonamide (DC_M01_12)
Synthesized by following general procedure A: Yellow solid (199 mg, yield 55%): m.p. 197-200 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.20-8.10 (m, 2H), 8.01 (d, J = 8.2 Hz, 1H), 7.98 -7.40 (m, 2H),
7.83-7.79 (m, 2H), 7.79-7.71 (m, 2H), 7.58-7.51 (m, 1H), 7.49-7.44 (m, 1H), 7.09 (q, J = 8.1 Hz, 4H),
3.96 (d, J = 6.2 Hz, 2H), 2.24 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 165.8, 153.4, 140.9, 138.9,
136.3, 135.5, 134.4, 134.2, 128.7, 128.1, 127.6, 127.0, 126.6, 125.7, 124.4, 122.7, 122.3, 45.9, 20.6.
ESI-MS m/z: 435 [M - H]-. ESI-HRMS calcd for C23H19N2O3S2 [M - H]- 435.0843, found 435.0834.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N,N-diethyl-2-fluorobenzenesulfonamide (DC_M01_13)
Synthesized by following general procedure B: Yellow solid (75 mg, yield 60%): m.p. 180-185 °C. 1H
NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 8.2 Hz, 2H), 8.12-8.08 (m, 1H),
8.06-8.05 (m, 1H), 7.98-7.87 (m, 2H), 7.61-7.55 (m, 1H), 7.54-7.48 (m, 1H), 3.25 (q, J = 7.0 Hz, 4H),
1.09 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 164.6, 153.3, 140.2, 137.4, 134.6, 130.9,
129.5, 128.9, 128.4, 126.9, 126.0, 124.2, 123.1, 122.4, 42.0, 14.2. ESI-MS m/z: 391 [M + H]+.
ESI-HRMS calcd for C19H20FN2O2S2 [M + H]+ 391.0945, found 391.0946.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-2-chloro-N,N-diethylbenzenesulfonamide (DC_M01_14)
Synthesized by following general procedure B Yellow solid (86 mg, yield 55%): m.p. 168-171 °C. 1H
NMR (500 MHz, DMSO-d6) δ 8.16-8.13 (m, 2H), 8.05-7.99 (m, 2H), 7.96-7.92 (m, 1H), 7.89 (d, J =

ACCEPTED MANUSCRIPT
16.2 Hz, 1H), 7.74 (d, J = 16.2 Hz, 1H), 7.58-7.52 (m, 1H), 7.51-7.48 (m, 1H), 3.34 (q, J = 7.1 Hz,
4H), 1.07 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 165.4, 153.4, 140.9, 137.5, 134.4,
133.9, 131.4, 131.3, 130.6, 126.7, 126.3, 125.9, 125.8, 122.8, 122.3, 41.2, 13.9. ESI-MS m/z: 407 [M
+ H]+. ESI-HRMS calcd for C19H20ClN2O2S2 [M + H]+ 407.0649, found 407.0647.
(E)-4-(2-(benzo[d]thiazol-2-yl)vinyl)-N,N-diethyl-2-methylbenzenesulfonamide (DC_M01_15)
Synthesized by following general procedure B: Yellow solid (80 mg, yield 54%): m.p. 140-145 °C. 1H
NMR (500 MHz, DMSO-d6) δ 8.15-8.12 (m, 1H), 8.04-8.00 (m, 1H), 7.88-7.86 (m, 1H), 7.84-7.76 (m,
3H), 7.69 (d, J = 16.2 Hz, 1H), 7.57-7.53 (m, 1H), 7.49-7.45 (m, 1H), 3.28 (q, J = 7.1 Hz, 4H), 2.57 (s,
3H), 1.07 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 165.8, 153.4, 139.1, 138.6, 137.3,
135.3, 134.2, 131.6, 129.2, 126.6, 125.7, 125.3, 124.5, 122.7, 122.3, 40.8, 19.8, 13.8. ESI-MS m/z:
387 [M + H]+. ESI-HRMS calcd for C20H23N2O2S2 [M + H]+ 387.1195, found 387.1192.
Molecular docking
All the available crystal structures of hMOF (2GIV, 2PQ8, 2Y0M, 2Y0N, 3QAH, 3TOA, 3TOB,
4DNC, 5J8C, 5J8F) were superimposed and the local flexibility of the residues around the cofactor
COA were observed when we checked the aligned structures. In order to rescue the poorly scored
compounds from standard virtual docking studies, Induced Fit Docking (IFD) protocol was selected to
predict the biding mode of the investigated compounds with relatively high activity against hMOF.
The X-ray structure of hMOF with both substrates and high resolution (PDB code 2GIV) among these
determined hMOF X-ray structures was fetched from PDB Web site and was prepared using default
protocol of the Protein Preparation Wizard (Schrödinger, LLC, New York, NY, 2015) from the
Maestro interface (Maestro, version 10.4; Schrödinger, LLC, New York, NY, 2015). Water molecules
and other HET atoms were deleted. The structures of investigated compounds were built by 2D
sketcher of Maestro, then these 2D structures were prepared by LigPrep (LigPrep, version 3.6;
Schrödinger, LLC: New York, NY, 2015), and their protonation states were generated at pH 7.0 ± 2.0
using Epik (Epik, version 3.4; Schrödinger, LLC: New York, NY, 2015). The binding site of initial

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glide docking was defined by the centroid of the ALY and the inner- and outer-box dimensions were
10 Å × 10 Å × 10 Å and 20 Å × 20 Å × 20 Å, respectively. An energy window of 2.5 kcal/mol was
applied for ring conformations sampling of the investigated compounds; conformations including
nonplanar feature of amide bonds were penalized. When the initial docking (Standard Precision Mode)
was performed using Glide software (Glide, version 6.9; Schrödinger, LLC: New York, NY, 2015),
side-chains of residues within a shell of 5 Å around each ligand were refined and optimized by Prime
(version 4.2, Schrödinger, LLC, New York, NY, 2015). Then investigated compounds were redocked
into the lowest energy structure within 30 kcal/mol. Top 20 poses were exported for each ligand for
visual inspection and further analysis.
Colony formation assay
HCT116 cells were plated in 6-well plates in a volume of 2 mL and treated with compounds in
corresponding concentration (DMSO as control) for 14 days. The supernatant was removed and the
cells were washed using PBS buffer. Then the colonies were stained by 0.1% crystal violet for 30 min.
The number of colonies were counted.
Western Blot Analysis
Total cell lysates were separated by 4%-16% SDS-polyacrylamide gels and transferred to
nitrocellulose membranes. The blots were blocked with blocking buffer (5% nonfat milk in PBST) for
30-60 min at room temperature and incubated with primary antibodies overnight at 4°C. Then the
blots were washed three times with PBST and incubated with 1:10000 dilution of donkey anti-rabbit
secondary antibody (HRP conjugated) for 1 h. Followed by another three washes, bands were detected
in a ChemiScope3400 imaging system using ECL substrate (Clinx).
Quantitative real time PCR
Total RNA was isolated from cells using TRIzol Reagent (Life Technologies) following the
manufacturer's instructions. cDNA was obtained by reverse transcription using HiScript II Q RT
SuperMix (Vazyme Biotech). QRT-PCR was performed using AceQ qPCR SYBR Green Master Mix
(Vazyme Biotech) and detected by Quant Studio 6 Flex Real-Time PCR system (ABI). GAPDH was
used as an internal control. Fold change of gene expression data was calculated by using of the ∆∆Ct

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= ∆Ct (GENE-GAPDH)normal – ∆Ct (GENE-GAPDH)cancer method. All Samples were run in
triplicates and results were presented as mean ± SD. Detailed sequence of the primers used in the
experiments can be found in the Supporting Information.
ASSOCIATED CONTENT
Supporting Information
The process of setting up AlphaScreen assay, the compound with high activity in second round
screening, the enzymatic selectivity of compound DC_M01_7, the result of MG149 in radioactive
acetylation assay, the cell viability assay of DC_M01_7 and MG149, primers used in qRT-PCR, the
1
spectral and analytical data of all compounds and copies of H NMR and 13C NMR spectra of all
compounds are in supporting in information.
AUTHOR INFORMATION
Corresponding Authors
*H.L.: telephone, 86-21-50806600; e-mail, hliu@simm.ac.cn
*C.L.: telephone, 86-21-50806600; e-mail, cluo@simm.ac.cn.
Author Contributions
#R.Z., J.W. contributed equally to this work.
ACKNOWLEDGMENTS
This work was supported by the Major Project of Chinese National Programs for Fundamental
Research and Development (2015CB910304), the National Natural Science Foundation of China
(21472209, 81625022, 21472208, and 81430084), and Chinese Academy of Sciences
(XDA12020353)
ABBREVIATIONS USED
µ,
micro;
µM,
micromole
per
liter;
°C,
degrees
celsius;
HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IC50, half-maximum inhibitory concentration;
IPTG, isopropyl β-D-1-thiogalactopyranoside; Kd, equilibrium dissociation constant; LB,
Luria−Bertani; mM, millimole per liter; min, minute; nM, nanomole per liter
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Figure 1. DC_M01 can inhibit hMOF in vivo and also can directly bind to hMOF. (A) The hit
DC_M01 was measured by radioactive assay on hMOF with an IC50 of 40 µM. (B) The Kd of
DC_M01 was determined using SPR as 19 µM. (C) The chemical structure of MG149
Figure 2. Design novel hMOF inhibitors.

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Scheme 1. General synthetic route to the target compounds DC_M01-DC_M01_12.
Scheme 2. Synthetic routes to compounds DC_M01_13-DC_M01_15
Table 1. The structure and inhibitory activity of DC_M01 and its analogs
Inh%
Inh%
IC50
Compd.
R1
R2
R3
R4
(50 µM)
53
64
57
0
(100 µM)
84
(µM)
DC_M01
5-F
5-Cl
H
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
H
40
DC_M01_1
DC_M01_2
DC_M01_3
DC_M01_4
DC_M01_5
Et
89
30
5-Br
H
Et
82
30
5-Me
5-OMe
6-Br
H
Et
14
-
-
H
Et
14
5
13
H
Et
21
-
DC_M01_6 5,6-di-Me
H
Et
95
93
9
96
7.7
6
-
DC_M01_7
DC_M01_8
DC_M01_9
DC_M01_10
DC_M01_11
DC_M01_12
DC_M01_13
DC_M01_14
DC_M01_15
MG149
H
H
H
H
H
H
H
H
H
H
Et
100
14
H
Me
H
Ph
13
1
25
-
H
Bn
H
11
-
H
4-Me-PhCH2
H
17
0
33
-
H
4-OMe-PhCH2
H
28
-
F
Et
Et
Et
Et
Et
16
7
18
-
Cl
Me
12
-
Et
28
100
30
-
-
100
15

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Figure 3. DC_M01_7 IC50 on hMOF in radioactive competitive assay. (A) The IC50 of DC_M01_7
did not alter when changing the concentration of acetyl coenzyme A. (B) The IC50 of DC_M01_7
decreased when increasing the concentration of histone 4 peptide.
Figure 4. (A) The binding mode of DC_M01 in the active site of hMOF. (B) The binding mode of
DC_M01_6 in the active site of hMOF. (C) The binding mode of DC_M01_7 in the active site of
hMOF. (D) The binding mode of DC_M01_15 in the active site of hMOF. All figures were prepared
using PyMol (http://www.pymol.org/).

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Figure 5. (A-B) DC_M01_7 was diluted to different concentrations from 100 µM to 6.25 µM to treat
HCT116 cells. The same volume DMSO is used as control group. Colonies were counted and pictured.
The inhibition rate of DC_M01_7 was calculated and pictured.

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Figure 6. Effect of DC_M01_7 on H4K16 acetylation and related genes’ expression in HCT116. (A)
DC_M01_7 can dose-dependently inhibit the acetylation of H4K16 acetylation. (B) DC_M01_7 can
decrease the expression of SKP2 and UCP2 and elevate the expression of IFI16.

ACCEPTED MANUSCRIPT
HIGHLIGHT
ò
ò
ò
We identified a potent hMOF inhibitor with a new scaffold using high throughput screening.
The binding affinity of the hit compound DC_M01 was measured by SPR.
DC_M01_7, which was obtained by chemical modification, could inhibit hMOF activity in a
substrate competitive mode.
ò
DC_M01_7 could inhibit hMOF activity in HCT116 cells and regulate downstream genes.