Design, synthesis and algicides activities of thiourea derivatives as the novel scaffold aldolase inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.01.023

By using a new Fragment-Based Virtual Screen strategy, two series of novel FBA-II inhibitors (thiourea derivatives) were de novo discovered based on the active site of fructose-1, 6-bisphosphate aldolase from Cyanobacterial (CyFBA). In comparison, most of the N-(2-benzoylhydrazine-1-carbonothioyl) benzamide derivatives (L14～L22) exhibit higher CyFBA-II inhibitory activities compared to N-(phenylcarbamothioyl) benzamide derivatives (L1～L13). Especially, compound L14 not only shows higher CyFBA-II activity (K_i = 0.65 μM), but also exhibits most potent in vivo activity against Synechocystis sp. PCC 6803 (EC₅₀ = 0.09 ppm), higher (7-fold) than that of our previous inhibitor (EC₅₀ = 0.6 ppm). The binding modes of compound L14 and CyFBA-II were further elucidated by jointly using DOX computational protocol, MM-PBSA and site-directed mutagenesis assays. The positive results suggest that strategy adopted in this study was promising to rapidly discovery the potent inhibitors with novel scaffolds. The satisfactory algicide activities suggest that the thiourea derivatives is very likely to be a promising lead for the development of novel specific algicides to solve Cyanobacterial harmful algal blooms (CHABs).

Full text of DOI:10.1016/j.bmc.2019.01.023

Accepted Manuscript
Design, synthesis and algicides activities of thiourea derivatives as the Novel
Scaffold Aldolase Inhibitors
Shan Xiao, Lin Wei, Zongqin Hong, Li Rao, Yanliang Ren, Jian Wan, Lingling
Feng
PII:
S0968-0896(18)31044-7
https://doi.org/10.1016/j.bmc.2019.01.023
BMC 14715
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 November 2018
17 January 2019
22 January 2019
Please cite this article as: Xiao, S., Wei, L., Hong, Z., Rao, L., Ren, Y., Wan, J., Feng, L., Design, synthesis and
algicides activities of thiourea derivatives as the Novel Scaffold Aldolase Inhibitors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.023
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Design, synthesis and algicides activities of thiourea derivatives as the Novel Scaffold
Aldolase Inhibitors
Shan Xiao,‡ Lin Wei,‡ Zongqin Hong, Li Rao, Yanliang Ren and Jian Wan* and Lingling Feng
International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education,
Department of Chemistry, Central China Normal University, Wuhan 430079, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
By using a new Fragment-Based Virtual Screen strategy, two series of novel FBA-II
inhibitors (thiourea derivatives) were de novo discovered based on the active site of fructose-1,
6-bisphosphate aldolase from Cyanobacterial (CyFBA). In comparison, most of the N-(2-
benzoylhydrazine-1-carbonothioyl) benzamide derivatives (L14~L22) exhibit higher
CyFBA-II inhibitory activities compared to N-(phenylcarbamothioyl) benzamide
derivatives (L1~L13). Especially, compound L14 not only shows higher CyFBA-II activity
(Ki = 0.65 μM), but also exhibits most potent in vivo activity against Synechocystis sp. PCC
6803 (EC50=0.09 ppm), higher (7-fold) than that of our previous inhibitor (EC50=0.6 ppm). The
binding modes of compound L14 and CyFBA-II were further elucidated by jointly using DOX
computational protocol, MM-PBSA and site-directed mutagenesis assays. The positive results
suggest that strategy adopted in this study was promising to rapidly discovery the potent
inhibitors with novel scaffolds. The satisfactory algicide activities suggest that the thiourea
derivatives is very likely to be a promising lead for the development of novel specific algicides
to solve Cyanobacterial harmful algal blooms (CHABs).
Available online
Keywords:
Aldolases
Fragment-based Virtual Screen
thiourea
algicides activities
De novo Discovery
 Corresponding author. Tel.: +86-027-67862022; fax: +86-027-67862022; e-mail: renyl@mail.ccnu.edu.cn;jianwan@mail.ccnu.edu.cn
‡ These authors contributed equally to this study

1. Introduction
Cyanobacterial harmful algal blooms (CHABs) have been increasing in frequency all over the world.[1] Toxin-producing CHABs
have directly threatened to the human health, living resources, and water economies.[2-5] To control these CHABs, many approaches
included mechanical, physical/chemical, and biological controls have been used so far.[6] In comparison, chemical control was the most
effective method. Chemical control involves the use of compounds (copper compounds, chemical oxidants, and herbicides).[1, 7]
Especially, the compounds designed for the specific target of cyanobacteria provide us a useful pathway to the development of potential
algaecides. Previously, using the systematically structure-centric approach, several novel inhibitors of class II fructose-1, 6-
bisphosphate aldolase from Cyanobacteria (CyFBA-II) were designed and synthesized in our group.[8, 9] In contrast, the N-(2,5-Dioxo-
2,5-dihydro-1H-pyrrol-1-yl) benzamide derivatives exhibit higher inhibitory activities against Synechocystis sp. PCC 6803.[9]
However, the instability of those compounds limits their further applications. To address this concern, two series of novel scaffold
CyFBA-II inhibitors (thiourea derivatives) were de novo designed by using Fragment-Based Virtual Screen (FBVS) strategy developed
in our group based on the substrate site of CyFBA-II. The proposed binding model of novel inhibitors targeting into CyFBA-II were
systematically identified by integrating Docking-ONIOM-XO (DOX)[10] computational protocol, Molecular Mechanics combined with
the Poisson–Boltzmann and Surface Area continuum solvation (MM-PBSA),[11] chemical synthesis, and site-directed mutant assays.
2. Results and Discussion
2.1. Design of thioureas derivatives as novel CyFBA-II inhibitors
In previous work, we found that N-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) benzamide derivatives shown potent inhibition against
CyFBA-II.[8] However, these compounds appears to be unstable due to the hydrazone moiety. Recently, de novo design (i.e. FBVS) are
well-developed and mature technique for lead discovery as well as lead optimization.[12, 13] Some de novo design computational
methods have already been implemented in commercial or academic software, such as LUDI,[14] EA-inventor,[15] SPROUT,[16]
BUILDER,[17] PROLIGANDS,[18] LigBuilder[19] and LEA3D,[20] especially, many protocols have successfully applied to the
discovery of highly potent inhibitors.[21-23] However, most of the software mentioned-above were performed based on their specific
docking algorithm and scoring function. Our previous studies[8, 9, 24] suggested that Surflex-Dock,[25] evaluated by using an
empirical function ChemScore, was suitable for the discovery of novel FBA inhibitors, thus Surflex-Dock were used in the present
FBVS protocol to generate and score the conformations of fragments and resulting compounds based on the CyFBA-II active pocket.
To design the algaecides with desirable druglike properties, several rings and frameworks in FDA-approved drugs[26] were used as the
fragments dataset (Figure S1) for this FBVS protocol (Figure 1).
Surflex Dock
(Score > 4.0)
Distances of heavy
atoms > 0.8Å
~48
~2,100,000
Connected
Molecules
Fragments
Surflex Dock
Xscore
Cluster Analysis
(RMSD<1.2)
~3512
Leak-like Rules
Percerta@ACD/Labs
~10
Actived
Molecules
Potent hit
compounds
Solubility > 0.1 mg/mL
MW sort
Figure 1. Fragment-Based Virtual Screen (FBVS) protocol
The 3D-Conformation of CyFBA-II were constructed as our previous work.[8, 9] The Zinc ion (Zn2+) were derived from the
template structure (PDB ID: 3GAY). The Molecular Dynamics (MD) simulation of CyFBA-II and substrate (FBP) were performed
prior to the docking, as illustrated in Figure S2 and Figure S3 in supporting information. In MD procedure, the charge of Zn ion is set
as 2, the parameters of Zn ion is optimized by Pang group[27, 28] in Amber package. For the FBVS protocol, the fragments were
docked firstly into the active pocket of CyFBA-II by using Surflex-Dock. Twenty poses were generated for each fragment. Then, the
cluster analysis for each fragment were performed (RMSD < 1Å), and six conformations with high score (surflex-dock score/number of
atoms > 0.15) will be selected for the connection between fragments. Subsequently, about 3512 molecules were generated by using the
connection module developed by our group. After that, the building compounds will be docked again into the CyFBA-II using Surflex-
Dock, and the Xscore developed by Wang et al.,[29] were performed for all docking conformations. Then, top 500 compounds with
high average score (Ave_score) of protein-ligand given by X-score were selected for the further Lead-like evaluation by using
Percepta@ACDLabs software. At this step, about 174 reasonable compounds were remained. Finally, top 10 compounds with smaller
Molecular Weight (MW) and solubility larger than 0.1 mg/mL were selected for our further analysis. Top 10 potent hit compounds
generated by FBVS routing were listed in Table 1.
Table 1. The top 10 hit compounds generated by present FMVS protocol

*ID
Compounds
LogPa
2.96
H-Bondsb
Solubilitya
13.13
Avg scorec
6.12
N
N
N
N
H
372
8
COOH
299
453
4.00
3.23
2
6
8.35
6.35
6.21
6.16
N
H
HO
NH2
OH
OH
O
247
157
406
HO
0.94
0.82
0.99
9
9
0.73
0.44
0.29
6.09
6.18
6.09
N
N
N
N
OH
N
N
O
N
N
HO
N
N
8
6
N
N
N
N
N
N
OH
O
278
3.32
2.60
0.20
0.18
6.35
6.13
O
HO
O
N+
O
S
-O
435 (L1)
10
N
N
H
H
OH
H
H
N
154
245
2.48
2.45
10
10
0.16
0.16
6.19
6.22
O
N
S
O
2N
OH
H
H
O
N
N
S
OH
O
2N
According to the total number of HDonors&HAcceptors and synthesis step number, compound 435, which was re-named L1, was
finally selected to further investigation; The synthetic route were illustrated in Scheme 1, which has been documented by Rauf et al.[30]
R3
R1
R2
R3
R
O
O
2
O
S
S
NH2
KSCN
C
Cl
N
N
N
Me2CO
R4
R4
Me2CO
R4
H
H
R1
R1 = H, OH, Cl, NO2; R2=H, NO2, Br; R3=H, NO2; R4=H, CF3, CH3, Ph, NO2, Cl
Scheme 1. General synthetic route for compound L1~L13.
Fortunately, the inhibitory constant (Ki) of L1 against CyFBA-II was of 16.7 μM (Table 2), this result indicates the FBVS route
adopted in the present study seems to be reliable.
2.2. Structure-activity-relationship (SAR) analysis
To discover more potent CyFBA-II inhibitors, it is necessary to analyze the structure-activity-relationship (SAR). Firstly, we fixed
R2, R3 and R4 to Hydrogen, and introduced the Cl, NO2, and OH group into R1 position (Table 2). However, no remarkable CyFBA-II
inhibition (Inhibition Rate < 40%) were observed, such as compounds L2 ~ L4. In comparison, compound L4 exhibit higher inhibition
rate (37%) than those of L2 and L3. This indicate that hydroxyl group seems to be favorable in R1 position. Furthermore, R1, R3 and R4
were fixed to hydrogen, and R2 was replaced by OH (L5), Br (L6) and NO2 (L7), the results show that compound L7 have relatively
high inhibition rate (60%) compared with those of compounds L5 (22%) and L6 (42%). Taken together, we believe that OH at R1 and

NO2 at R2 position appears to be favorable. To explain this experiment observation, L11 was chosen as the representative compound,
the binding model of L11 and CyFBA-II was predicted by using the DOX strategy, which has been documented previously.[10] The
possible binding conformation of L11 and CyFBA-II was illustrated in Figure 2.
Table 2. Substitution patterns of compounds L1 – L13 and their inhibition activity against CyFBA-II
R4
R1
H
H
N
N
S
O
R2
R3
Ki (μM)/
Compds
L1
R1
R2
R3
R4
Inhibition Ratea
OH
NO2
H
H
16.7±0.4
30%a
L2
L3
Cl
H
H
H
H
H
H
H
H
H
H
H
H
NO2
OH
H
H
H
15%a
L4
H
H
37%a
L5
OH
Br
H
22% a
L6
H
H
42% a
L7
H
NO2
NO2
NO2
NO2
NO2
H
H
60% a
L8
OH
OH
OH
OH
OH
OH
CF3
CH3
Ph
NO2
H
5.5±0.10
5.5±0.20
2.5±0.10
1.9±0.02
9.1±0.20
9.1±0.10
L9
L10
L11
L12
L13
NO2
NO2
H
Cl
As shown in Figure 2, the hydroxyl group and sulfur of L11 can coordinate with the Zn2+. Moreover, the hydroxyl can also form
hydrogen bond with residue N275. To verify the importance of residue N275, the site-directed mutagenesis was performed. As listed in
Table 3, the inhibitory activity of L11 against N275A (37.9μM) decreased about 20-fold than parental CyFBA-II (1.9 μM), the possible
reason for this case is that N275A results in the disappear of the hydrogen-bond between N275 and the hydroxyl moiety in benzene ring
(Figure 2). Additionally, Figure 2 illustrate that the nitro group in R2 position can form hydrogen bonds with residues K202 and T278.
As expected, the inhibitions of L11 against T278A decreased about 46.3-fold than parental CyFBA-II (Table 3). Unfortunately, K202A
mutant lacked detectable enzymatically activity, thus the interaction of compound and K202 could not be determined using mutagenesis
experiment. Combing the DOX results (Figure 2), we believe that the interaction between nitro group in R2 position and T278 is
probably existed. Therefore, the compounds with hydroxyl group in R1 and NO2 group in R2 position could exhibit high inhibitory
activities against CyFBA-II.
Subsequently, we fixed R1 and R2 to OH and NO2, and tried to vary the R4 to CF3 (L8), CH3 (L9), Ph (L10) and NO2 (L11).
Compared with L1 (16.7 μM), L8 ~ L11 show higher inhibitions (Ki =1.9~5.5 μM) against CyFBA-II. Especially, the L11 exhibit
highest CyFBA-II inhibitions with Ki value of 1.9 μM. These findings reveal that the introduction of substituents in R4 position,
hydrophilic or hydrophobic group, might improve the CyFBA-II inhibitions of hit compounds. However, our DOX prediction (Figure
2) show that the nitro group in R4 position locate outside of the active pocket, no remarkable interactions of nitro group in R4 position
could be observed. Therefore, the reason that compounds L8~L11 have higher CyFBA-II inhibitions is not clear so far.

In addition, one can notice from Table 2 that L12 exhibit higher inhibitory activity (9.1 μM) compared with compound L4 (37%),
this indicate that the NO2 in R3 position seems to be favorable than H. The reason is likely that NO2 in R3 position could also form
hydrogen-bond with T278.
One can notice from Figure 2 that, the NH moiety in the middle linkage of L11 might form a hydrogen-bond with D277, and the
carboxyl group of L11 may form hydrogen-bond with residues R281 and R302. However, the D277A mutant couldn’t affect the
inhibitory activity of L11 (2.4-fold), this is not consistent with the predicted interaction in Figure 2, maybe due to the flexibility of
residue D277. In contrast, the mutants of R281A and R302A could result in 12.9 and 76-fold decrease of the L11 inhibitions (Table 3).
In addition, the Van der Waals interaction between thiourea linkage and R281 could also be observed in Figure 2. These may be the
partial reason that the inhibitions of L11 against R281A significantly decreased about 76-fold compared with parental CyFBA-II.
Figure 2. The predicted binding modes of compound L11 and CyFBA-II
2.3. Optimization of hit compounds.
Based upon the mutant data (Table 3), it is concluded that R281 is significantly important for the binding of L11 against CyFBA-II.
However, due to the effect of the coordination between sulfur and Zn2+, the hydrogen-bond distance of L11 and R281 is larger than 3 Å
(Figure 2). Thus, we propose that if one amide group were interposed between hydroxyl and sulfur, the hit compounds will be closer to
R281, which in turn improve the inhibitory activities of hit compounds against CyFBA-II. For this purpose, L14~L22 were further
synthesized (Table 4), the synthetic route was illustrated in Scheme 2.
Table 3. The Ki values of the compound L11 and L14 against wildtype (WT) and mutants of CyFBA-II
L11(µM)
L14 (µM)
Ki
Ki(mutant)/Ki(WT)
Ki
Ki(mutant)/Ki(WT)
WT
1.9±0.02
3.9±0.20
3.3±0.20
3.4±0.10
37.9±1.4
4.6±0.60
87.5±0.50
143.7±1.5
24.4±1.1
1
0.7±0.02
15.2±0.30
3.9±0.50
1.0±0.10
15.9±1.0
7.2±0.50
4.3±0.50
52.3±0.40
20.4±0.10
1
S50A
2.1
1.7
2.1
20
23.4
6.0
1.5
24.5
11
R51A
S55A
N275A
D277A
T278A
R281A
R302A
2.4
46.3
76.0
12.9
6.6
80.5
31

As listed in Table 4, most of these compounds have high inhibitory activities against CyFBA-II (Ki ≤
5.2 μM). When R1 was replaced by hydroxyl group, the compounds (L14~L16) exhibit higher inhibitory
activities against CyFBA-II (Ki =0.65~0.88μM). Especially, compound L14 exhibits highest inhibition with
Ki of 0.65 μM. These results indicate that the hydroxyl group in R1 position are significantly important for
the inhibitory activities of hit compounds. The possible binding modes (Figure 3) of compound L14 and
CyFBA-II were also predicted by using DOX strategy and MD simulation, the plots of MD were illustrated
in Figure S4.
R2
R1
O
R1
O
R3
R2
R3
NH2
N
O
S
O
O
H
H
S
N
KSCN
C
N
N
Cl
N
H
H
Me2CO
Me2CO
R4
R4
R4
R1=H, OH; R2=H, Br, NO2; R3=H, OH, F, Br, NO2; R4=H, F, Cl
Scheme 2. General synthetic route for L14~L22
Similar to L11, the hydroxyl group in R1 of L14 could form hydrogen bond with N275 (Figure 3). When
N275 was mutated to Ala, the inhibitions of L14 against N275A decreased about 25-fold compared to WT.
Therefore, we believe that the hydrogen-bond between N275 and L14 is essential for binding of L14 and
CyFBA-II (Figure 3).
Table 4. Substitution patterns of compounds L14 – L22 and their inhibition activity against CyFBA-II
and Synechocystis sp. PCC 6803
R2
R3
R1
O
S
O
H
N
N
N
H
H
R4
Compds
L14
R1
R2
R3
R4
Ki (μM)
EC50 (ppm)
OH
H
H
H
0.65±0.05
0.09±0.02
L15
L16
L17
L18
L19
L20
L21
L22
OH
OH
OH
H
H
H
Cl
F
0.63±0.03
0.88±0.03
1.25±0.16
1.87±0.09
3.1±0.17
3.5±0.71
5.2±0.10
2.8±0.12
0.98±0.34
5.36±0.73
6.4±0.80
0.39±0.13
5.9±1.1
H
H
H
OH
H
H
H
H
H
H
H
NO2
Br
H
H
H
H
F
8.2±0.60
1.58±0.20
16.3±6.2
H
H
Br
NO2
H
H
Additionally, one carbonyl group in the left of the thiourea linkage could coordinate with Zn2+, while the
other carbonyl group could form coordination with Zn2+ via water molecule (Figure 3). The remarkable
hydrogen bonds between thiourea linkage and surrounding residues (S50, R281 and R302) could be

observed. As expected, when the S50, R281 and R302 were mutated to Ala, the corresponding inhibitory
activities of L14 decrease about 23.4, 80.5, 31-fold (Table 3). This is satisfactory consistent with our
theoretical predictions that these three residues could form hydrogen bond with compound L14. Noticeably,
the R281A mutant could result in the 80-fold decrease of L14 inhibitory activities (Figure 3), this is owning
to the Van der Waals interaction between thiourea linkage and R281 and R302, which is similar to the
binding-modes of L11 and CyFBA-II (Figure 2).
It should be noticed that the NO2 moiety of L11 can form a hydrogen-bond with T278 (Figure 2), the
Ki of L11 against T278A is significantly increased 46.3-fold (Table 3) compared to parental CyFBA-II. In
contrast, no hydrogen-bond between L14 and D278 could be formed (Figure 3), thus the effect of D278A on
the inhibitory activity of L14 is slightly, with the decrease of 6.6-fold (Table 3). In addition, our DOX&MD
results show no interaction between L14 and residues R51 and S55. As expected, when S55, R51 were
mutated, the corresponding Ki value decreased less than 10-fold. The well agreement between experimental
and theoretical results show that our predicted interactions between L14 and CyFBA-II is reasonable.
Figure 3. The possible binding modes of L14 and CyFBA-II obtained by DOX strategy and molecular dynamic (MD)
simulations
The R4 substituent of hit compound located out of the active cavity, therefore, our substituent in R4
position actually couldn’t improve the inhibition activities of hit compounds, such as L14~L16.
Furthermore, the binding free energies of L14 and the mutants (N275A, R281A, S50A and R302A) were
also calculated by using MMGBSA method based on the MD results. Our MMGBSA results (Table 5) show
that the R281A and R302A could result in the remarkable decrease (6.29 and 6.21 kcal/mol) of ΔΔGbind
while R275A and S50A mutants could lead to relatively small decrease (5.99 and 3.44 kcal/mol) of ΔΔGbind
,
.
These are qualitatively agreement with the experimental Ki value. Taken together, we could believe that the
binding modes of L14 and CyFBA-II predicted by DOX and MD were reasonable, and R281 maybe a
critical pharmacophore to the binding of hit compounds.
2.4. Synechocystis Activity
The inhibition activities (EC50) of compounds L14~L22 against Synechocystis sp. PCC 6803 were further
determined (Table 4). As listed in Table 4, most compounds with high CyFBA-II inhibition activities
generally exhibit potent Synechocystis activities. For example, the inhibition constant (Ki) of compounds
L14 ~ L22 were less than 6 μM, correspondingly, the EC50 of most of these compounds is less than 10 ppm.
Especially, compound L14 shows highest CyFBA-II inhibition activity (0.65μM), and also exhibits most
potent algicide activity (EC50 = 0.09 ppm) which is 7-fold potent than our previous inhibitor (Y10) with EC50
of 0.6 ppm.[24] However, it is hard to make direct correlations between CyFBA-II inhibitions and algicide
activities from the currently available experimental data, because the absorption, distribution, and
metabolism performance of individual compound obtained in the present study. For instance, the CyFBA-II
inhibitory activities of compounds L16 (0.88μM), L17 (1.25μM) and L22 (2.7μM) is higher, but these
compounds show relatively weak algicide activities, with the EC50 of 5.36 ppm, 6.4 ppm, and 16.3 ppm,
respectively.

Table 5. Binding free energy of compound L14 and mutant and wild-type (WT) enzyme of CyFBA-II
calculated by MMGBSA, together with experimental Ki values
ΔGbing(kcal/mol)
ΔΔGbind(kcal/mol) Ki(μM)
Ki(mutant)/Ki(WT)
WT
-30.56
0
0.65±0.02
1
N275A
R281A
S50A
-24.57
-24.27
-27.12
-24.35
5.99
6.29
3.44
6.21
15.93±1.0
52.34±0.41
15.2±0.29
20.44±0.14
24.5
80.5
23.4
31.0
R302A
To further confirm that the algicide activities of these hit compounds is achieved by inhibiting CyFBA-II
enzyme, the inhibition of the representative compound L14 against overexpressed Synechocystis sp. PCC
6803 (PSBII), whose FBA-II gene was overexpressed (Figure 4B). Due to the increase of FBA-II gene in
Synechocystis sp. PCC 6803, the algicide activity of L14 against PSBII (EC50=4.98μM, Figure 5B) was
decreased about 18-fold compared to the activity of L14 against WT Synechocystis sp. PCC 6803
(EC50=0.28μM). The result demonstrated that L14 could actually inhibit CyFBA-II in Synechocystis sp.
PCC 6803, in turn inhibit the growth of the Cyanobacterial.
100
120
(A)
(B)
100
80
60
40
20
0
80
60
40
20
0
EC50=0.28±0.06μM
EC50=4.98±0.46μM
-5
0
5
10
15
20
25
30
35
-20
0
20
40
60
80
100 120 140 160 180
L14 (WT)
L14 (PSBII)
Figure 4. The inhibition curve of compound L14 against Synechocystis sp. PCC 6803. (A) Synechocystis sp. PCC 6803 from
wild-type (WT) strains; (B) Synechocystis sp. PCC 6803 from CyFBA-II overexpression strains (PSBII).
3. Conclusions
Herein, using a new Fragment-Based Virtual Screen (FBVS) strategy developed in our group, two series
of novel FBA-II inhibitors (thiourea derivatives) were de novo discovered based on the active site of
fructose-1, 6-bisphosphate aldolase from Cyanobacterial (CyFBA). In comparison, most of the N-(2-
benzoylhydrazine-1-carbonothioyl) benzamide derivatives (L14~L22) exhibit higher CyFBA-II inhibitory
activities compared to N-(phenylcarbamothioyl) benzamide derivatives (L1~L13). When the OH was
introduced on the benzoylhydrazine moiety of the benzamide derivatives, the corresponding compounds,
such as L14(0.65μM), L15(0.63μM) and L16(0.88 μM), exhibit high CyFBA-II inhibitory activities.
Furthermore, the algicide activities were determined for Synechocystis sp. PCC 6803. The results suggest
that most of the compounds with high CyFBA-II inhibition might exhibit potential in vivo activities.
Especially, compound L14 not only shows high CyFBA-II activities (Ki = 0.65 μM), but also exhibits
highest algicide activities (EC50 = 0.09 ppm), which is 7-fold potent than our previous inhibitor (EC50=0.6
ppm). The possible interactions of representative compound L14 and surrounding key residues in the active
site of CyFBA-II were elucidated by jointly using DOX, MD simulations, MM-PBSA and Site-directed
mutagenesis assays. The well agreement between theoretical and experimental results suggest that the DOX
strategy is a reliable approach for the prediction of the binding-model of inhibitor and target, and the present
design strategies were successfully implemented to rapidly screen and optimization of novel inhibitors. The
satisfactory algicide activities suggest that the thiourea derivatives are very likely to be a promising lead
compound for the development of novel specific algicides to solve CHABs.
4. Experimental
4.1. Molecular dynamics (MD) simulations and Binding Free Energies

To verify the binding mode of hit compounds and CyFBA-II, the MD simulations were further performed
by using pmemd.cuda module of AMBER16. The hit compounds were optimized by Gaussian 09 at the
ωb97xd/6-31g(d) level, and the whole protein was optimized using Amber force field. The AMBER10 force
field and gaff force field were used for CyFBA-II and inhibitors, respectively. The whole system was
immersed with TIP3P water molecules[31] in a truncated octahedron box of 10 Å from any solute atoms.
The system was neutralized with the counterions of Na+. SHAKE[32] was used to constrain bonds involving
hydrogen atoms, and the time step was 2.0 fs. the long-range electrostatics were calculated by the particle
mesh Ewald (PME) algorithm.[33] The nonbonded cutoff was set to 10 Å. Based on the MD trajectories, the
binding free energies L14 were also calculated using MM/GBSA. The electrostatic desolvation free energy
was calculated by the Generalized Born (GB) model.[34] The binding interface of CyFBA-II (or it’s mutant)
and inhibitor is relatively hydrophobic. The solvent and the solute dielectric constants of GB were set to 80
and 1, respectively.[35] The nonpolar desolvation term was estimated from the solvent accessible surface
area (SASA): 0.0072 × SASA + 0.00.[36]
4.2. Chemicals and Reagents.
The proton (1H) and carbon (13C) NMR spectra were recorded on a Varian Mercury-Plus 400 or 600 MHz
spectrometer in CDCl3, DMSO-d6 or acetone-d6. Chemical shifts were given in parts per million (ppm) with
TMS as the internal reference. Flash chromatography purifications were performed on Merck silica gel 60
(230−400 mesh) as the stationary phase, and MeOH and CH3COCH3 were used as eluents. High-resolution
mass spectrometry (HRMS) analysis was performed using a hydride IT-TOF mass spectrometer with ESI
interface (Shimadzu, Kyoto, Japan) and Agilent 6224 Accurate-Mass time-of-flight mass spectrometer with
1
ESI interface (Agilent Technologies, Waldbronn, Germany). The synthetic procedure, H and 13C nuclear
magnetic resonance (NMR) and high-resolution mass specitrometry (HRMS) are listed in the Supporting
Information.
4.3. Enzyme inhibition activities and Site-Directed Mutagenesis.
Triosephosphate isomerase (TIM), glycerol 3-phosphate dehydrogenase (GPDH), and FBA-I were from
rabbit muscle and purchased from Sigma Corporation. The inhibition constant (Ki) of the hit compounds
were determined at the CyFBA-II (or it’s mutant) recombinant protein level as described previously.[37] The
recombinant protein was expressed in E. coli BL21(DE3) cells, it was purified as previous document.[9, 37]
Commercial preparations from Sigma of glycerol 3-phosphate dehydrogenase (GPDH) from rabbit muscle
and triosephosphate isomerase (TIM) from rabbit muscle were used. The inhibitory activity of hit
compounds against CyFBA-II were determined by using an NADH linked enzymatic assay.[38, 39] The
activity of cleavage reaction was examined in Tris-HCl buffer (50mM, pH 7.5), NADH (0.6 mM), glycerol
3-phosphate dehydrogenase (GPDH, 0.3 U, Sigma), triosephosphate isomerase (TIM, 1 U, Sigma), aldolase
(1.4 μg/mL), and various concentrations of fructose-1, 6-bisphosphate (FBP, 0.04‒5mM) in a cuvette to give
the final volume of 0.4 mL. The reaction progress was measured by monitoring the change in absorbance of
NADH at 340 nm on the microplate reader (SpectraMax M5, Molecular Devices) over 5 min at 30 °C. The
dose-response curves of compounds L10, L11, L14, L15 and L16 against wild-type CyFBA-II were
illustrated in Figure S5.
Ki values were determined by the inhibition of the reaction at more than two concentrations of the
substrate (FBP). In present study, The L11 and L14 were selected for the validation of the competitive
inhibitors, as shown in Figure S6. For the competitive inhibitors, Ki may be calculated by the equation[40]:
Ki = IC50/(1+S/Km), where IC50 is the half-maximal inhibitory concentration, S is the concentration of FBP
and Km is the Michaelis-Menten constant. The Km value of CyFBA-II is 68 µM. The inhibition rate (I%) was
calculated using the following formula: I% = [(V0−V)/V0] × 100%, where V0 represent the maximum velocity
([I] = 0 μM), and V indicate the velocity when an inhibitor was added. All data were analyzed by GraphPad
Prism 6.0 software (Prism Software).
Mutations were performed by introducing specific base changes into a double-stranded DNA plastid. All
highly effective site-directed mutagenesis were performed using KODPlus Mutagenesis kit (Toyobo, Osaka,
Japan) in term of the manufacturer’s protocol as our previous document.[41] The wild-type CyFBA-II
plasmid and mutagenic primers were denatured, annealed, and polymerized in a final volume of 50 μL for 25
cycles (95 °C for 30s, 60 °C for 1 min, and 72 °C for 3 min). Additionally, Nde I and BamH I restriction
enzyme (Fermentas, China) was used to digest the parental methylated and hemi-methylated DNA. Then, the
plasmids of mutations were transformed into Escherichia coli (E. coli) strain BL21 (DE3) cells, and the
sequence analysis were performed for validations. By using SDS-PAGE, the identical motilities were
exhibited for all purified enzymes (wild and mutants) to ensure the purities more than 90%.
4.4. Inhibitory Assays on Synechocystis sp. PCC 6803.
Synechocystis sp. PCC 6803 were cultured photoautotrophically in BG11 medium[42] at 28 °C, it is in
light for 12 h and in dark for 12 h alternatively, and 50−55% relative humidity for seven days in 96-well

microtiter plates as described previously.[9] This is similar to the antibacterial bioassay.[43] The inhibition
rate (I%) of test compounds on the cyanobacteria growth was determined according to previous work.[8]
The half maximal effective concentration (EC50) values were obtained from the inhibition curves for the
inhibition rate against various concentrations of hit compounds. The curves were fitted by nonlinear
regression using logistic equation in Origin 7.7 software.
Acknowledgments
This work was supported by the Natural Science Foundation of China (Nos. 21877046, 21873035,
21572077 and 21472061), the Program for the PCSIRT (No. IRT0953), and the self-determined research
funds of CCNU from the colleges’ basic research and operation of MOE (Nos. CCNU18TS010,
CCNU18ZDPY02, CCNU16A02041 and CCNU14A05006), the support from the Program of Introducing
Talents of Discipline to Universities of China (111 Program, B17019) is also appreciated.
Supplementary Material
Experimental procedures, analytical data, enzyme assays, and biological evaluation. This material can be
found online
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Design, synthesis and algicides activities of thiourea
derivatives as the Novel Scaffold Aldolase Inhibitors
Shan Xiao, Lin Wei, Zongqin Hong, Yanliang Ren*, Li Rao, Lingling Feng and Jian Wan*
International Cooperation Base of Pesticide and Green Synthesis (Hubei), Key Laboratory of Pesticide & Chemical
Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079,
China