Design, Synthesis, and Potency of Pyruvate Dehydrogenase Complex E1 Inhibitors against Cyanobacteria

Article abstract of DOI:10.1021/acs.biochem.7b00636

Safe and effective algaecides are needed to control agriculturally and environmentally significant algal species. Four series (6, 10, 17, and 21) of 29 novel 4-aminopyrimidine derivatives were rationally designed and synthesized. A part of 10, 17, and 21 displayed potent inhibition of Escherichia coli pyruvate dehydrogenase complex E1 (E. coli PDHc-E1) (IC₅₀ = 2.12-18.06 μM) and good inhibition of Synechocystis sp. PCC 6803 (EC₅₀ = 0.7-7.1 μM) and Microcystis sp. FACH 905 (EC₅₀ = 3.7-7.6 μM). The algaecidal activity of these compounds positively correlated with their inhibition of E. coli PDHc-E1. In particular, 21l and 10b exhibited potent algaecidal activity against PCC 6803 (EC₅₀ = 0.7 and 0.8 μM, respectively), values that were 2-fold increased compared to that of copper sulfate (EC₅₀ = 1.8 μM), and showed the best inhibition of cyanobacterium PDHc-E1 (IC₅₀ = 5.10 and 6.06 μM, respectively). 17h and 21e, the best inhibitors of E. coli PDHc-E1, were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. These results revealed that the improved inhibition of novel inhibitors compared with that of the lead compound I was due to the formation of a new hydrogen bond with Leu264 at the active site of E. coli PDHc-E1. The results proved the great potential to obtain effective algaecides via the rational design of PDHc-E1 inhibitors. [Figure Presented]

Full text of DOI:10.1021/acs.biochem.7b00636

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Article
Design, Synthesis and Potency of Pyruvate Dehydrogenase
Complex E1 Inhibitors against Cyanobacteria
Yuan Zhou, Jiangtao Feng, Hongwu He, Leifeng Hou, wen
jiang, Dan Xie, Lingling Feng, Meng Cai, and Hao Peng
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00636 • Publication Date (Web): 09 Oct 2017
Downloaded from http://pubs.acs.org on October 11, 2017
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Design, Synthesis and Potency of Pyruvate Dehydrogenase Complex
E1 Inhibitors against Cyanobacteria
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Yuan Zhou , Jiangtao Feng , Hongwu He , Leifeng Hou, Wen Jiang, Dan Xie,
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Lingling Feng, Meng Cai, Hao Peng
College of Chemistry, Central China Normal University; Key Laboratory of Pesticide
& Chemical Biology, Ministry of Education, 152 Luoyu Road, Wuhan, 430079,
China.
*
Corresponding author: (Tel: +86ꢀ27ꢀ67867958; Fax: +86ꢀ27ꢀ67867960; Eꢀmail:
he1208@mail.ccnu.edu.cn; penghao@mail.ccnu.edu.cn)
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These authors equally contributed to this work.
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ABSTRACT: Safe and effective algaecides are needed to control agriculturally and
environmentally significant algae species. Four series (6, 10, 17, and 21) of 29 novel
4ꢀaminopyrimidine derivatives were rationally designed and synthesized. A part of 10,
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7, and 21 displayed potent inhibition against Escherichia coli pyruvate
dehydrogenase complex E1 (E. coli PDHcꢀE1) (IC50 = 2.12ꢀ18.06 ꢁM) and good
inhibition against Synechocystis sp. PCC 6803 (EC = 0.7ꢀ7.1 ꢁM) and Microcystis
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sp. FACH 905 (EC50 = 3.7ꢀ7.6 ꢁM). The algaecidal activity of these compounds
positively correlated with their inhibnitio against E. coli PDHcꢀE1. In particular, 21l
and 10b exhibited potent algaecidal activity against PCC 6803 (EC50 = 0.7 and 0.8
ꢁM, respectively), which were 2ꢀfold increase on the potency if compared to copper
sulfate (EC50 = 1.8 ꢁM), also showed the best inhibition against cyanobacteria
PDHcꢀE1 (IC50 = 5.10 and 6.06 ꢁM, respectively). 17h and 21e with the best
inhibition against E. coli PDHcꢀE1 were studied by molecular docking, siteꢀdirected
mutagenesis and enzymatic assays. These results revealed that the improved
inhibition of novel inhibitors compared with the lead compound I was due to forming
new hydrogen bond with Leu264 at the active site of E. coli PDHcꢀE1. The results
proved a high potential to obtain effective algaecides by rational design of PDHcꢀE1
inhibitors.
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Cyanobacterial harmful algal blooms (CyanoꢀHABs) are one of the serious
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consequences of eutrophication in many parts of the world. Such blooms pose a
serious water pollution problem that impacts human health, living resources, and
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water safety. Various approaches have been developed to deal with HABs, which
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can be classified as mechanical, physical, chemical, and biological controls.
Chemical control is regarded as one of the most effective methods against
CyanoꢀHABs. Chemical control involves use of herbicides, copperꢀbased compounds,
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and chemical oxidants. At present, copper sulfate, copper oxychloride, and copper
citrate are the most commonly used algaecides. It is noteworthy that the application of
these compounds containing copper can lead to the accumulation of cupric ion in
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water, which would result in ecological risks. In addition, several herbicides, such as
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diquat, paraquat, atrazine, and simazine have been used as effective algaecides.
However, these herbicides were nonꢀspecific and could increase cyanotoxins in the
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water. Moreover, some chemical oxidants, such as hydrogen peroxide, chlorine
dioxide, calcium peroxide, and potassium permanganate are also used as potential
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algaecides due to their fast effect and good sustainability.
However the effect of
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some oxidants as specific algaecides need to be further evaluated.
Although these chemicals could act as effective algaecides, none of them was
specifically designed targeting the enzymes of cyanobacteria or developed as an
exclusive algaecide. Thus, it is an interesting and valuable research topic to design
specially effective and safe algaecide. Recently, the computer aided virtual screening
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for bioactive pharmacophores has been proved to be successful for some protein
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targets.
Therefore, the computer aided design of new inhibitors toward critical
enzyme of bacteria or cyanobacteria may provide useful solution for the development
of potential algaecides.
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The pyruvate dehydrogenase complex (PDHc) in microorganisms plays a vital
role in cellular metabolism and catalyzes the oxidative decarboxylation of pyruvate
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and the subsequent acetylation of coenzyme A (CoA) to acetylꢀCoA.
The overall
reaction of oxidative decarboxylation was simply shown in Figure 1. PDHc contains
three enzymatic components including pyruvate dehydrogenase (E1),
dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and a
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number of cofactors.
The pyruvate dehydrogenase complex E1 component
(PDHcꢀE1) catalyzes the first step of the multistep processes, using thiamine
2+
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diphosphate (ThDP) and Mg as cofactors. Particularly, this PDHcꢀE1 catalyzed
process is a rate limiting step among multistep processes. PDHcꢀE1 is, therefore, a
viable target to discover novel algaecides.
Figure 1. Oxidative decarboxylation catalyzed by PDHc.
To the best of our knowledge, the crystal structure of PDHcꢀE1 from
cyanobacteria has not been reported. At the moment it is difficult to perform the
structureꢀbased rational design of novel inhibitors against PDHcꢀE1 from
cyanobacteria directly. However, the crystal structure of E. coli PDHcꢀE1 has been
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determined. On that basis, we built an optimal binding model for inhibitors into
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active site of PDHcꢀE1 from E. coli using molecular simulation. In recent years, a
systematic study for the design and synthesis of inhibitors against E. coli PDHcꢀE1
was carried out in our laboratory. A series of 2ꢀmethylpyrimidineꢀ4ꢀylamine
derivatives as ThDP analogs, such as I, II, and III (Figure 2) had been chemically
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synthesized and demonstrated to be effective inhibitors against E.coli PDHcꢀE1.
Some of the compounds, by the modification of I, exhibited moderate to good
30,31
inhibitory activity against both E. coli PDHcꢀE1 and cyanobacteria.
The results
showed that the inhibitory potency of some compounds against cyanobacteria was
related to their inhibition against E.coli PDHcꢀE1. On the other hand, the
cyanobacteria, which is similar to bacteria, belong to prokaryotes. They all have
cytoplasm, ribosome and nucleoid. We thought that it is possible to obtain an effective
algaecide by designing potent PDHcꢀE1 inhibitors with the binding model of active
site of E. coli PDHcꢀE1. Therefore, we tried to design more potent PDHcꢀE1
inhibitors by the modification of I with the aid of E. coli PDHcꢀE1 structureꢀbased
molecular docking methods. Our previous studies have shown that the
2
ꢀmethylpyrimidineꢀ4ꢀylamine and 1,2,3ꢀtriazole ring is a pharmacophore acting on E.
coli PDHcꢀE1. Therefore, the moieties of 2ꢀmethylpyrimidineꢀ4ꢀylamine and
1,2,3ꢀtriazole ring were kept in structure I, and further optimization was focused on
the modification of substituted phenoxy moiety. The analysis of molecular docking
indicated no interaction between oxygen atom of phenoxy moiety and residues in the
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active site of PDHcꢀE1. It is expected that the inhibition would be enhanced by
introducing a heterocyclic ring to replace phenoxy moiety in structure I.
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Figure 2. Structures of ThDP and known PDHcꢀE1 inhibitors.
Therefore, the phenoxy moiety in I was replaced with tetrahydrophthalimide
moiety, which is a structural unit of fungicide captan (Figure 3), to give the new
compound 6. As a comparison, compound 10a was designed by introducing a
phthalimide moiety into I to replace the phenoxy moiety. To explore the effect of
different heterocycles on E. coli PDHcꢀE1 inhibition, compound 17 was designed by
replacing the phthalimide moiety in 10a with the benzotriazinone moiety.
Furthermore, in order to improve the inhibition against both E. coli PDHcꢀE1 and
cyanobacteria, a better hydrophobic group quinazolinone moiety (LogP: 0.98) was
introduced by replacing the benzotriazinone moiety (LogP: 0.84) of 17 to give the
new compound 21. Meanwhile, the study of structureꢀactivity relationship (SAR) of I
indicated that both inhibitory activity against E. coli PDHcꢀE1 and antifungal activity
could be further increased by introduction of iodine at the 5ꢀposition of
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,2,3ꢀtriazole. Therefore, iodine atom was also introduced in a series of compounds
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0, 17, and 21.
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Figure 3. Design of title compounds.
Herein, four series (6, 10, 17, and 21) which included 29 4ꢀaminopyrimidine
derivatives were rationally designed and synthesized. The interaction between
inhibitors and E. coli PDHcꢀE1 was studied by molecular docking and siteꢀdirected
mutagenesis. The inhibitory activities against E. coli PDHcꢀE1 and two common
algae species (Synechocystis sp. PCC 6803 and Microcystis sp. FACH 905) were
tested. Some representative compounds were further examined their inhibitory
activity against cyanobacteria PDHcꢀE1. To find safe inhibitors for mammals, the
enzymeꢀselectivity of representative compounds between porcine and E. coli
PDHcꢀE1 was also examined.
MATERIALS AND EXPERIMENTAL DETAILS
Chemicals Characterization and Reagents. Melting points (m.p.) were
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measured on an electrothermal melting point apparatus and were uncorrected. H
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NMR and F NMR spectra were recorded on a Bruker spectrometer at 400 and 376
MHz, respectively, with deuterated dimethyl sulfoxide (DMSOꢀd ) as the solvent and
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TMS as the internal standard. Chemical shifts are reported in δ parts per million
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(
ppm). Highꢀresolution electron impact mass spectra (HRꢀEIMS) were recorded under
electron impact (70 eV) conditions using a MicroMass GCT CA 055 spectrometer.
Elemental analysis (EA) was measured on a Vario ELIII CHNSO elemental analyzer.
Crystal structure was recorded by Bruker AEPEX DUO CCD diffractometer.
Unless otherwise noted, reagents were purchased from commercial suppliers and
used without further purification. Column chromatography purification was carried
out using silica gel. ThDP, pyruvate, and porcine PDHcꢀE1 were purchased from
SigmaꢀAldrich.
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Synthesis Procedures
General
procedure
for
preparation
of
5-azidomethyl-2-methyl
pyrimidine-4-ylamine (1).
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Compound 1 was prepared according to the procedure previously reported. To
a solution of Vitamin B1 (thiamine chloride) (10.3 g, 30.6 mmol) and sodium azide
(4.9 g, 75.4 mmol) in water (100 mL) was added sodium sulfite (0.38 g, 3.0 mmol)
o
and the mixture was stirred at 65 C for 5 h. Citric acid (21.0 g, 100 mmol, to pH ≈ 4)
was added and the aqueous solution was washed with dichloromethane. Potassium
carbonate (to pH ≈ 8) was added, upon which some precipitation of the product
occurred. The suspension was filtered and the filtrate was extracted with ethyl acetate
and the combined organic layers were washed with brine, dried with MgSO and
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evaporated under reduced pressure. The solid residue was pooled with the precipitate
and recrystallized from ethyl acetateꢀhexane to give the azide (1) as fine needles in 60%
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yield.
General procedure for preparation of N-iodomorpholine (3).
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Compound 3 was prepared according to the procedure previously reported. To
a stirred solution of iodine (12.7 g, 0.05 mol) in 200 mL of methanol was added
dropwise morpholine 2 (4.35 g, 0.05 mol). The reaction mixture was stirred at 20ꢀ25
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C for 1 h. The mixture was then filtered and washed with methanol to obtain orange
crystals in 75ꢀ80% yield.
General procedure for preparation of alkynes (5 and 8).
3
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Compound 5 was prepared according to the procedure previously reported. To
a solution of tetrahydrophthalimide 4 (1.51 g, 10 mmol) in acetone (20 mL),
potassium carbonate (2.76 g, 20 mmol) and propargyl bromide (1.43 g, 12 mmol)
were added sequentially. The resulting mixture was refluxed for 8 h. After cooling to
room temperature, water (50 mL) was added, which was extracted with EtOAc (100
mL) for 3 times. The organic layer was washed with brine (50 mL), dried over
2 4
anhydrous Na SO , filtered and concentrated to give compounds (5). Under this
condition, 8 was also prepared.
General procedure for preparation of iodoalkynes (9).
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Compound 9 was prepared according to the procedure previously reported. To
a solution of terminal alkyne 8 (2 mmol) in THF (10 mL) was added
Nꢀiodomorpholine (3) (1.36 g, 4 mmol), followed by CuI (0.04g, 0.2 mmol). The
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reaction mixture was stirred at 20ꢀ25 C for 4ꢀ6 h. The reaction mixture was filtered.
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The filtrate was evaporated under reduced pressure to afford yellow solid 9, which
were used directly without further purification.
General procedure for synthesis of the 2-methylpyrimidine-4-ylamine derivatives
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and 10.
CuI (0.04 g, 0.2 mmol) was added to
5ꢀazidomethylꢀ2ꢀmethylpyrimidineꢀ4ꢀylamine (1) (0.33 g, 2 mmol) and alkynes 5 (2
mmol) in THF (10 mL) followed by Et N (0.24 g, 2.4 mmol). After overnight stirring
a
stirred solution of
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at room temperature, the reaction mixture was poured into water (30 mL). The
precipitate was collected by filtration and dried under atmospheric pressure. The
crude product was further purified by recrystallization (methanol/dichloromethane) to
give the title compound 6. Under this condition, 10 were also prepared.
General procedure for preparation of isatoic anhydride (12).
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Compound 12 was prepared according to the procedure previously reported. A
mixture of anthranilic acid 11 (50 mmol) and tetrahydrofuran (THF, 100 mL) was
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stirred at ꢀ10 C for 30 min. Then, a solution of triphosgene (BTC, 50 mmol) in THF
(30 mL) was added dropwise to the above mixture. After that, the mixture was stirred
o
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for 1 h at ꢀ10 C, followed by 18ꢀ24 h at 20ꢀ25 C. The solvent was removed under
reduced pressure, and anhydrous ether (150 mL) was added to the obtained residue
with vigorous stirring. The precipitate was collected by filtration, washed with
anhydrous ether, and dried to give compound 12 in yields of 80%ꢀ90%.
General procedure for preparation of anthranilamide (13).
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Compound 13 was prepared according to the procedure previously reported. A
suspension of isatoic anhydride 12 (40 mmol), ammonium carbonate (160 mmol), and
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1,4ꢀdioxane (150 mL) was heated at 60 C. After stirring for 5ꢀ8 h, the reaction
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mixture was cooled to room temperature and evaporated under reduced pressure, and
then water (200 mL) was added to the residue, which was extracted with EtOAc (300
mL ) for three times. The organic layer was washed with brine (100 mL), dried over
anhydrous Na
0ꢀ85%.
General procedure for preparation of 1,2,3-benzotriazin-4-ones (14).
2 4
SO , filtered and concentrated to afford compound 13 in yields of
8
38
Compound 14 was prepared according to the procedure previously reported. A
solution of sodium nitrite (4.14 g, 60 mmol) in 0.5 N HCl (240 mL) was stirred at 0
o
C for 20 min. Anthranilamide 13 (30 mmol) dissolved in N,Nꢀdimethylformamide
(DMF, 15 mL) was then added dropwise to the above solution for 40 min. After
o
another 1 h of stirring at 0 C, 30% aqueous ammonia was added slowly to adjust the
pH to 10.0. The reaction mixture was allowed to stir for 15 min and then reacidified to
pH 2.0. After stirring for 30 min, the precipitated product was filtered off with suction,
washed with water (200 mL), and dried to afford compound 14 in yields of 75ꢀ83%.
General procedure for preparation of alkynes (15) and iodoalkynes (16).
Compounds 15 and 16 were prepared according to the procedures described for 5
and 9, respectively.
General procedure for synthesis of the 2-methylpyrimidine-4-ylamine derivatives
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1
1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
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4
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5
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5
5
5
5
5
5
5
5
6
containing 1,2,3-benzotriazin-4-ones moiety 17.
CuI (0.04 g, 0.2 mmol) was added to
a
stirred solution of
5ꢀazidomethylꢀ2ꢀmethylpyrimidineꢀ4ꢀylamine (1) (0.33 g, 2 mmol) and alkynes 15 or
iodoalkynes 16 (2 mmol) in THF (10 mL) followed by Et N (0.24 g, 2.4 mmol). After
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
overnight stirring at room temperature, the reaction mixture was poured into water (30
mL). The precipitate was collected by filtration and dried under atmospheric pressure.
The
methanol/dichloromethane) to give the title compound 17.
General procedure for preparation of 4(3H)-quinazolinones (18).
crude
product
was
further
purified
by
recrystallization
(
39
Compound 18 was prepared according to the procedure previously reported. A
mixture of anthranilic acid 11 (0.1 mol) and formamide (18 g, 0.4 mol) was heated at
o
1
30ꢀ135 C. After stirring for 4 h, water (40 mL) was added to the mixture. The
o
reaction mixture was cooled to 60 C, water (20 mL) was added to the mixture. After
stirring for 30 min, the precipitated product was filtered off with suction. The crude
products were recrystallized with ethanol to give compound 18 in yields of 80ꢀ95%.
General procedure for preparation of alkynes (19) and iodoalkynes (20).
Compounds 19 and 20 were prepared according to the procedures described for 5
and 9, respectively.
General procedure for synthesis of the 2-methylpyrimidine-4-ylamine derivatives
containing quinazolinone moiety 21.
CuI (0.04 g, 0.2 mmol) was added to
a
stirred solution of
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Biochemistry
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2
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5
6
7
8
9
5
ꢀazidomethylꢀ2ꢀmethylpyrimidineꢀ4ꢀylamine 1 (0.33 g, 2 mmol) and 19 or 20 (2
3
mmol) in THF (10 mL) followed by Et N (0.24 g, 2.4 mmol). After overnight stirring
at room temperature, the reaction mixture was poured into water (30 mL). The
precipitate was collected by filtration and dried under atmospheric pressure. The
crude product was further purified by recrystallization (methanol/dichloromethane) to
give title compounds 21.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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34
35
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Crystallographic Study
The crystal of the title compound 21f was cultured from the mixture of methanol
and dichloromethane. Xꢀray singleꢀcrystal diffraction data for 21f were collected on a
Bruker AEPEX DUO CCD diffractometer at 298(2) K using Mo Kα radiation (λ =
0.71073 Å) by
π and ω scan mode. The program SAINT+ was used for integration of
the diffraction profiles. The structure was solved by the direct method using the
SHELXS program of the SHELXTL package and refined by fullꢀmatrix leastꢀsquares
4
0
methods with SHELXL. All nonꢀhydrogen atoms of 21f were refined with
anisotropic thermal parameters. All hydrogen atoms were placed in geometrically
idealized position and constrained to ride on their parent atoms.
Enzymatic Inhibition Activity and Half Maximum Inhibition Concentrations
(IC50)
To evaluate the inhibition of title compounds against E. coli PDHcꢀE1, IC were
50
determined. The cloning, expression, purification and activity of E. coli PDHcꢀE1
25
were carried out according to the reported method. Enzymatic activities were
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2
2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
5
measured according to the published method. For IC50 determination, a standard
reaction mixture containing 50 mM PO (pH 6.4), 0.4 mM
K
3
4
2,6ꢀdichlorophenolindophenol (DCIP), 50 ꢁM sodium pyruvate as the substrate, 5 ꢁg
PDHcꢀE1 purified enzyme and 50 ꢁM ThDP. Title compounds in concentrations
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
ranging from 0 to 200 ꢁM were incubated for 5 min with E. coli PDHcꢀE1 at 37 C
prior to addition of the substrate pyruvate. The IC50 values were determined by
nonlinear leastꢀsquares fitting of the data using the Hill kinetic equations in the
41
Growth/sigmoidal model from origin 7.0 software as described previously.
Inhibitory Assays (in vivo) on Cyanobacteria
Synechocystis sp. PCC 6803 and Microcystis sp. FACH 905 were cultured
42
o
photoautotrophically in BG11 medium at 28 C for 7 days at a 12:12 h of light/dark
43
cycle at 50ꢀ55% relative humidity. The inhibition of title compounds against
cyanobacteria growth was calculated according to the equation: I = {1ꢀ[∆ꢀꢁꢂ,ꢃꢄꢅ
∆ꢀꢁꢆ,ꢃꢄꢅ]/∆ꢀꢁꢇ,ꢃꢄꢅ}× 100%, where, I is the relative inhibition (%); ∆ꢀꢁꢇ,ꢃꢄꢅ and
ꢀꢁꢂ,ꢃꢄꢅ are the average data of optical densities at 680 nm for the cyanobacteria in
−
∆
the blank control and treatment, respectively; and ∆ꢀꢁꢆ,ꢃꢄꢅ is the average data
optical density at 680 nm for the test compound in each assay. When cyanobacteria
growth was significantly inhibited, the maximum half effective concentrations (EC50
)
were estimated with logistic fitting. The widely used algaecide copper sulfate was the
positive control, while DMSO was the solvent control. Each test was measured in
triplicate.
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6
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8
9
Inhibition against Porcine PDHc-E1
To test the inhibitory activity against porcine PDHcꢀE1, the relative inhibition (%)
of title compounds were assayed at 100 ꢁM. The total volume of 100 ꢁL reaction
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
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43
44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
mixture contained 1.0 mM MgCl
2
,
0.2 mM ThDP,
0.5 mM
3
ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT), 6.5 mM
phenazine methosulfate (PMS), 50 mM K
3
PO
4
, pH 7.0, 5 ꢀL enzyme and 100 ꢁM
o
compound. The reaction mixture was incubated for 3 min at 37 C, then added sodium
pyruvate (5 ꢁL, 40 mM) to initiate reaction. The optical density at 566 nm (OD566
)
o
was recorded. The reaction mixture was then incubated for 5 min at 37 C, and the
OD566 was recorded. The relative inhibition was calculated according to the equation:
I= [(∆ꢀꢁꢈ,ꢉꢃꢃ − ∆ꢀꢁꢂ,ꢉꢃꢃ)/∆ꢀꢁꢈ,ꢉꢃꢃ]ꢊ× 100%, where, I is the relative inhibition (%);
∆
∆
ꢀꢁꢈ,ꢉꢃꢃ is the average data of optical density at 566 nm in the blank control; and
ꢀꢁꢂ,ꢉꢃꢃ is the average data of optical density at 566 nm in the presence of
compound. Each test was measured in triplicate.
Molecular Docking
The binding modes of selected compounds were investigated using an AutoDock
44
4
.2 program. The threeꢀdimensional (3D) structure of the PDHcꢀE1 was obtained
from PDB database (PDB code: 1L8A). Hydrogens were added to the protein
45
structure by pdb2pqr server. The structure of coꢀcrystal ligand ThDP and
compounds (Ic, 10a, 17h and 21e) were respectively prepared with SYBYL7.0 and
Gasteiger charges were used for these inhibitors. The original ligand ThDP was first
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1
1
1
1
1
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1
1
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2
2
2
2
2
2
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3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
docked into the binding site. In the docking process, the Lamarckian genetic
algorithm (LGA) was applied for the conformational search of the binding complex of
ThDP with PDHcꢀE1. The grid size was set to be 60×60×60 and the used grid space
was the default value of 0.375 Å, other parameters were set to default. Among a set of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
00 conformational candidates of the docked complex structures, the topꢀranked one
was selected according to the interaction energy. The predicted binding mode of
ThDP was compared with the conformation of ThDP extracted from the crystal
structure. The same docking parameters were subsequently adopted to perform
molecular docking of Ic, 10a, 17h and 21e, respectively.
Site-Directed Mutagenesis of PDHc-E1
Siteꢀdirected mutagenesis of PDHcꢀE1 was accomplished by the introduction of
specific base changes into a doubleꢀstandard DNA plasmid according to reported
41
method.
DNA encoding of the wildꢀtype PDHcꢀE1 cloned into the
pMALꢀC2xꢀPDHcꢀE1 was used as a template for mutagenesis. The standard PCR
mixture contained 50ꢀ100 ng of template DNA and 100ꢀ200 ng of each mutagenizing
primer. The methylated plasmid was digested with DpnI, and 4 ꢁL of each reaction
was used to transform the DH5α competent cells. All mutations were confirmed by
DNA sequencing. Verified plasmids containing the desired mutations were
transformed into the E.coli TB1 strain. The mutant PDHcꢀE1 proteins were purified
by the same manner as the purification of wildꢀtype PDHcꢀE1.
RESULTS AND DISCUSSION
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Biochemistry
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6
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8
9
Synthesis
A total of 29 new compounds in the series of 6, 10, 17, and 21 was designed and
synthesized (Scheme 1, 2, and 3). The synthetic route is simple and convenient.
In this synthetic route, 5ꢀazidomethylꢀ2ꢀmethylpyrimidineꢀ4ꢀylamine (1) that
was prepared starting from thiamine hydrochloride (Vitamin B1) is a key intermediate
in the synthesis of 6, 10, 17, and 21. The skeleton of all title compounds 6, 10, 17, and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
1 contain 1,2,3ꢀtriazole ring. The 1,2,3ꢀtriazole ring in the skeleton of title
compounds could be readily constructed by applying ‘click chemistry’. In the present
work, 6 or 10 was synthesized via 1,3ꢀdipolar cycloaddition reaction employing
copper(I) iodide and triethyl amine from 1 by reacting with terminal alkynes 5, 8 or
iodoalkynes 9, which was prepared by the reaction of propargyl bromide and
tetrahydrophthalimide 4 or phthalimide 7 (Scheme 1).
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9
1
1
1
1
1
1
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1
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1
2
2
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2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthetic route for the title compounds 6 and 10.
The title compound 17 was further synthesized by this Cuꢀcatalyzed 1,3ꢀdipolar
cycloaddition reaction of 1 with 15 or 16, which was prepared by the reaction of
1
,2,3ꢀbenzotriazinꢀ4ꢀones 14 with propargyl bromide (Scheme 2). 14 was prepared
from anthranilamides 13 through the combination of diazotization, nucleophilic
addition, and cyclization in one pot. 13 was prepared starting from anthranilic acids
11, which was converted to the desired isatoic anhydride 12 via the annulation
reaction with BTC. Then, 12 reacted with ammonium to give the anthranilamide 13.
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Biochemistry
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2
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5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthetic route for the title compound 17.
The title compound 21 was also synthesized by 1 reacting with 19 or 20 via
1
,3ꢀdipolar cycloaddition in the presence of CuI and Et N (Scheme 3). 19 was
3
prepared from the key intermediate 4(3H)ꢀquinazolinone 18, which was prepared by
the reaction of 11 with formamide.
Scheme 3. Synthetic route for the title compound 21.
In this synthetic method, iodine atom was introduced in title compounds by using
iodoalkynes 9, 16 or 20, which was prepared via 8, 15 or 19 reacting with
Nꢀiodomorpholine 3, respectively, under the catalysis of copper(I) iodine. The title
compounds and CuI were both hard to dissolve in water and most organic solvent. It
was difficult to separate them by using a column chromatographic method. However,
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
it was found that the mixed solvent (methanol/dichloromethane) could increase the
solubility of the title compounds, but CuI could not dissolve in the mixed solvent.
Therefore, the crude product was dissolved in the hot mixed solvent and immediately
filtered to remove CuI. The filtrate was cooled and filtered to give the desired title
compounds.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The abovementioned synthetic method has the advantage of being simple, mild,
1
19
easy, and high yield. Those compounds were characterized with H NMR, F NMR,
HRMS spectra and confirmed by elementary analysis. The characterization data were
listed in the Supporting information.
The Crystal Structure of Compound 21f
The crystal data of 21f were presented in Table 1, and Figure 4 gives a
46
perspective view of 21f with the atomic labeling system. This result indicates that
2
1f has a “U” shape conformation, which is close to its binding pose.
Figure 4. Crystal structure of compound 21f by Xꢀray diffraction determination.
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9
Table 1. Crystallographic data of compound 21f.
Parameters
Data
chemical formula
formula weight
crystal system
space group
crystal color
a (Å)
C H BrN O
17
15
8
427.26
monoclinic
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C2/c
colorless
15.235(4)
b (Å)
16.300(4)
c (Å)
17.449(5)
α
β
γ
(deg)
(deg)
(deg)
90
108.188(4)
90
V (Å)
Z
4116.5(19)
8
ꢀ3
Dcalc (Mg⋅m )
1.482
θ
range (deg)
hkl range
F (0 0 0)
1.99ꢀ25.50
ꢀ18 ≤ h ≤ 18; ꢀ19 ≤ k ≤ 19; ꢀ21 ≤ l ≤ 21
1872
no. collected refl.
14767
no. ind. Refl. (Rint
)
3812 (0.1054)
3812 / 2 / 286
2.029
data/ restraints/ parameters
ꢀ1
Absorption coefficient (mm )
R1; > 2 )]
R1; (all data)
GOOF
w
R
2
[
I
σ(I
0.0745; 0.1828
0.1751; 0.2412
0.995
wR
2
In vitro Inhibition against E. coli PDHc-E1
Compounds 6 and 10 were first synthesized and evaluated for their inhibition
26
against E.coli PDHcꢀE1. The IC50 values, including Ia-b, were summarized in Table
2
. It was very interesting to find that 10a (IC50 = 9.46 ± 0.52 ꢁM) and 10b (IC50 = 5.96
0.06 ꢁM) with a phthalimide moiety showed promising inhibitory activity, which
±
had 6ꢀfold and 9ꢀfold activity higher than that of Ia, respectively. However, 6 with a
tetrahydrophthalimide moiety showed very weak inhibitory activity. The reason for
this is that the phthalimide moiety of 10 could form πꢀπ interaction with His142
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
according to the analysis of molecular docking (Figure 5B). This result indicated that
phthalimide moiety is a very important pharmacophore. Meanwhile, 10b with iodine
1
as R exhibited better enzyme inhibitory activity than that of corresponding
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
compound 10a with H as R .
Table 2. Structures and IC50 values against E.coli PDHcꢀE1 of compounds I, 6, and
10.
1
a
Compd.
R
IC50 (ꢁM)
Ia
Ib
6
H
I
55.15±4.65
19.56±0.00
b
H
H
I
NT
9.46±0.52
5.96±0.06
1
0a
10b
a
IC50 (ꢁM) value is defined as the micromolar concentration required for 50%
inhibition against PDHcꢀE1 from E. coli in vitro
.
b
NT: not tested. The primary inhibitory potency was less than 100% at 100 ꢁM.
For exploring the effect of different heterocycles on E.coli PDHcꢀE1 inhibition,
1
7 was designed and synthesized by replacing the phthalimide group with
benzotriazinone moiety. The IC50 values of 17 were summarized in Table 3. The
series 17 showed moderate to good inhibition with IC50 values in the range of
1
1
8.06ꢀ3.72 ꢁM. Most of 17 with iodine as R showed better enzyme inhibition than
1
1
2
that of corresponding compounds 17 with H as R , such as 17b (R = H, R = 6ꢀCl,
1
2
1
IC50 = 18.06 ± 0.91 ꢁM) < 17h (R = I, R = 6ꢀCl, IC50 = 3.72 ± 0.20 ꢁM), 17f (R = H,
2
1
2
R = 8ꢀCH , IC = 11.25 ± 0.44 ꢁM) < 17l (R = I, R = 8ꢀCH , IC = 5.19 ± 0.26
3
50
3
50
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ꢁ
M). Among these compounds, 17h showed the best inhibition (IC50 = 3.72 ± 0.20
M). This result proved that the inhibitory activity against E. coli PDHcꢀE1 could be
ꢁ
further increased by introducing iodine at the 5ꢀposition of 1,2,3ꢀtriazole.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Structures and IC50 values against E.coli PDHcꢀE1 of compounds I, 17 and
2
1.
1
2
a
1
2
a
Compd.
R
R
IC50 (ꢁM) Compd.
R
R
IC50 (ꢁM)
Ic
H
H
H
H
H
H
H
I
4ꢀCl
7ꢀCl
6ꢀCl
5ꢀCl
6ꢀBr
5ꢀF
26.44±1.68
6.88±0.38
18.06±0.91
7.05±0.08
6.33±0.43
7.18±0.31
11.25±0.44
4.08±0.20
3.72±0.20
13.35±0.54
6.70±0.34
8.29±0.48
5.19±0.26
6.69±0.10
21b
21c
21d
21e
21f
21g
21h
21i
H
H
H
H
H
H
H
I
7ꢀNO
7ꢀCl
6ꢀCl
5ꢀCl
6ꢀBr
5ꢀF
2
3.44±0.20
6.87±0.17
6.54±0.20
2.12±0.05
4.29±0.17
19.97±0.59
23.92±0.75
4.55±0.20
10.36±0.30
4.71±0.14
8.21±0.26
8.67±0.31
9.81±0.17
1
7a
7b
17c
7d
7e
17f
7g
7h
17i
7j
7k
1
1
1
8ꢀCH
7ꢀCl
6ꢀCl
5ꢀCl
6ꢀBr
5ꢀF
3
6,7ꢀOCH
7ꢀCl
3
1
1
I
21j
21k
21l
21m
21n
I
6ꢀCl
I
I
I
I
I
I
5ꢀCl
6ꢀBr
1
1
8ꢀCH
6,7ꢀOCH
3
3
1
7l
I
8ꢀCH
8ꢀCH
3
3
I
2
1a
H
a
IC50 (ꢁM) value is defined as the micromolar concentration required for 50%
inhibition against PDHcꢀE1 from E. coli in vitro
.
The improved inhibition of 17 from the lead structure I motivated further
optimization by replacing the benzotriazinone moiety in structure 17 with a
quinazolinone moiety. The inhibitory potency of 21 was slightly improved (Table 3),
1
2
1
compared with 17, such as 21e (R = H, R = 5ꢀCl, IC = 2.12 ± 0.05 ꢁM) > 17c (R
50
2
1
2
=
H, R = 5ꢀCl, IC50 = 7.05 ± 0.08 ꢁM), 21d (R = H, R = 6ꢀCl, IC50 = 6.54 ± 0.20
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
2
ꢁ
M) > 17b (R = H, R = 6ꢀCl, IC50 = 18.06 ± 0.91 ꢁM). 21e (IC50 = 2.12 ± 0.05 ꢁM)
showed the best inhibition in series 21. It was noticed that compounds with
2
electronꢀwithdrawing groups on the benzene ring as R seem to be more effective than
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
these compounds with electronꢀdonating alkyl counterparts as R .
In vivo Inhibition against Cyanobacteria
In this test, copper sulfate was used as a positive control. Copper sulfate is a
commonly used effective algaecide against cyanobacteria. However, it is also against
nonꢀtarget organisms, and harmful to ecosystems due to its heavy metal ions. Most of
the compounds exhibited excellent activity against the cyanobacteria PCC 6803 and
FACH 905 (Table 4). The inhibitory potency against cyanobacteria is related to the
inhibition against E. coli PDHcꢀE1 (Tables 2, 3, and 4). Generally, compounds with
higher E. coli PDHcꢀE1 inhibition displayed potent inhibition against cyanobacteria.
For instance, 10, 17, 21a-f, and 21i-n had higher E. coli PDHcꢀE1 inhibition (IC50
2.12ꢀ18.06 ꢁM) and also exhibited potent inhibitory potency against PCC 6803 (EC50
0.7ꢀ7.1 ꢁM) and FACH 905 (EC50 = 3.7ꢀ7.6 ꢁM). 21l and 10b, particularly,
=
=
exhibited excellent algaecide activity against PCC 6803, which was 2ꢀfold better than
copper sulfate (EC50 = 1.8 ꢁM). In comparison, I, 6, and 21g-h with lower inhibition
(
IC50 > 19 ꢁM) against E. coli PDHcꢀE1 also displayed significantly lower algaecide
activities (EC > 50 ꢁM).
5
0
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6
7
8
9
Table 4. Structures and in vivo EC50 values against cyanobacteria of compounds I, 6,
1
0, 17, and 21.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
a
EC50 (ꢁM) inhibitory
EC50 (ꢁM) inhibitory
1
2
1
2
Compd.
R
R
against cyanobacteria
PCC6803 FACH905
Compd.
R
R
against cyanobacteria
PCC6803 FACH905
Ia
Ib
Ic
H
I
H
H
>50
>50
17l
21a
21b
21c
21d
21e
21f
I
8ꢀCH
8ꢀCH
3
3
5.6±0.8
4.6±0.5
5.0±0.1
4.7±0.2
5.3±0.4
3.9±0.1
4.4±0.3
>50
6.5±0.5
6.6±0.6
7.4±0.4
7.1±0.5
7.6±0.7
5.7±0.6
5.7±0.4
>50
>50
>50
H
H
H
H
H
H
H
H
I
H
4ꢀCl
>50
>50
7ꢀNO
7ꢀCl
6ꢀCl
5ꢀCl
6ꢀBr
5ꢀF
2
6
>50
>50
10a
H
I
2.6±0.1
0.8±0.1
5.5±0.8
6.2±0.9
7.1±0.7
5.9±0.8
6.9±0.2
5.8±0.8
6.2±0.6
4.8±0.4
5.1±0.3
5.5±0.6
4.6±0.5
4.4±0.2
4.1±0.1
5.7±0.3
5.7±0.3
6.8±0.4
5.8±0.4
7.3±0.6
5.8±0.2
4.6±0.4
3.7±0.3
4.9±0.3
6.6±0.5
4.3±0.3
1
0b
7a
1
H
H
H
H
H
H
I
7ꢀCl
6ꢀCl
5ꢀCl
6ꢀBr
5ꢀF
17b
7c
7d
17e
7f
7g
17h
7i
7j
7k
21g
21h
21i
1
6,7ꢀOCH
3
>50
>50
1
7ꢀCl
2.5±0.2
2.8±0.1
2.5±0.1
0.7±0.1
2.6±0.1
2.8±0.1
1.8±0.1
4.1±0.1
4.4±0.2
4.0±0.1
4.4±0.2
4.3±0.1
4.4±0.1
1.5±0.1
21j
I
6ꢀCl
1
8ꢀCH
7ꢀCl
6ꢀCl
5ꢀCl
6ꢀBr
5ꢀF
3
21k
21l
I
5ꢀCl
1
I
6ꢀBr
I
21m
21n
I
8ꢀCH
3
1
I
I
6,7ꢀOCH
3
1
I
CuSO
4
1
I
a
The inhibitory activity against synechocystis sp. PCC 6803 and Microcystis sp.
FACH 905.
6
with a tetrahydrophthalimide moiety exhibited much weak inhibition against
both E. coli PDHcꢀE1 and cyanobacteria (Tables 2 and 4). However, 10a with a
phthalimide moiety exhibited prominent activities against both E. coli PDHcꢀE1 and
cyanobacteria due to the πꢀπ interaction between His142 and phthalimide moiety
1
based on molecular docking (Figure 5B). When iodine as R was introduced into the
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2
3
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6
7
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
structure of 10a to give 10b, it was found that 10b showed more powerful inhibitory
activity (EC50 = 0.8 ꢁM) against PCC 6803 than that of 10a and control.
It was further testified that inhibition against cyanobacteria could be remarkably
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
enhanced by introducing iodine at the 5ꢀposition of 1,2,3ꢀtriazole of structure 21
1
(
Table 4). All 21 with iodine as R showed higher inhibitory activity against
1
2
cyanobacteria than that of these corresponding compounds with H as R . When R
kept same, 21n (IC50 = 9.81 ± 0.17 ꢁM) and 21i (IC50 = 4.55 ± 0.20 ꢁM) with iodine
1
as R exhibited higher inhibition against E. coli PDHcꢀE1 than that of 21h (IC50
=
1
2
3.92 ± 0.75 ꢁM) and 21c (IC = 6.87 ± 0.17 ꢁM) with H as R , respectively (Tables
50
3
and 4). Meanwhile, 21n (EC50 = 4.4ꢀ2.8 ꢁM) and 21i (EC50 = 4.1ꢀ2.5 ꢁM) with
1
iodine as R also exhibited higher inhibitory activity against both PCC 6803 and
1
FACH 905 than that 21h (EC50 > 50 ꢁM) and 21c (EC50 = 7.1ꢀ4.7 ꢁM) with H as R ,
respectively. The results showed that it is possible to obtain an effective algaecide by
designing a potent E.coli PDHcꢀE1 inhibitor.
Enzyme-Selective Inhibition
Although some reported analogs of ThDP exhibited highly inhibitory activity
against E. coli PDHcꢀE1, they had poor enzymeꢀselective inhibition between
mammals and microorganisms. For example, reported compound ThTTDP (Figure 2)
exhibited potent inhibition against E. coli PDHcꢀE1 (Ki = 64 nM), however, it also
24,47
displayed powerful binding to human PDHcꢀE1 (
K
i
= 74 nM).
Therefore, it is
important to find selective PDHcꢀE1 inhibitors safe to mammals. In this work,
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2
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5
6
7
8
9
ThTTDP was used as a positive control. 17f, 21a, and 21m with good inhibition
against both E. coli PDHcꢀE1 (IC50 = 5.19ꢀ8.67 ꢁM) and cyanobacteria were chosen
to test their inhibition activity against porcine PDHcꢀE1. These compounds showed
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
00% inhibition against E. coli PDHcꢀE1 at 100 ꢁM, but negligible inhibition (<30%)
against porcine PDHcꢀE1 (Table 5). Moreover, they all exhibited fairly low inhibition
than that of ThTTDP (IC50 = 2.00±1.45 ꢁM). Which means these designed
compounds could selectively inhibit E. coli PDHcꢀE1, may have low mammalian
toxicity.
Table 5. Inhibition of 17f, 21a, and 21m against E. coli and porcine PDHcꢀE1.
E. coli PDHcꢀE1
IC50 (ꢁM) Inhibitory potency (%)
Porcine PDHcꢀE1
Compd.
a
b
b
Inhibitory potency (%)
1
7f
1a
1m
5.19±0.26
6.69±0.10
8.67±0.31
100.00±0.57
100.00±0.89
100.00±0.63
28.79±0.43
23.94±1.29
21.67±0.32
2
2
1
00.00±0.37
c
ThTTDP
2.23±0.56
100.00±0.15
(
IC50 = 2.00±0.45 ꢁM)
a
IC50 (ꢁM) value is defined as the micromolar concentration required for 50%
inhibition on PDHcꢀE1 from E. coli in vitro
.
b
Inhibitory potency (%) of compounds against enzyme in vitro at 100 ꢁM as average
of triplicate.
c
ThTTDP: Thiamin thiothiazolone pyrophosphate.
Analyses of the Interaction
To understand the binding model of novel inhibitors (10, 17 and 21) with E. coli
PDHcꢀE1, the representative compounds 10a, 17h, and 21e and Ic (as a comparison)
were performed computational simulation studies (Figure 5).
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7
8
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1
1
1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Optimal binding model for compounds Ic (A), 10a (B), 17h (C), and 21e (D)
at the active site of E. coli PDHcꢀE1 docked by Autodock 4.2. PDHcꢀE1 is shown as
ribbon, ligands and some key residues are shown as stick, and hydrogen bonds are
shown as dashed lines.
1
0a, 17h, and 21e can occupy the ThDPꢀbinding pocket, and bind in the active
site of E. coil PDHcꢀE1 with the ‘V’ conformation. On the right side of the ‘V’
conformation, the 4ꢀaminopyrimidine ring of 10a, 17h or 21e form hydrogen bonds
with Met194, Glu571 and Val192, respectively, which is similar to the interactions of
2
4
ThDP or Ic and corresponding residues. On the left side of the ‘V’ conformation,
similar to Ic, there is a πꢀπ interaction between His142 and the heterocycle moiety of
1
0a, 17h or 21e, respectively.
Comparing with lead compound Ic (IC50 = 26.44 ± 1.68 ꢁM), the inhibition of
0a (IC = 9.46 ± 0.52 ꢁM), 17h (IC = 3.72 ± 0.20 ꢁM), or 21e (IC = 2.12 ± 0.05
1
50
50
50
ꢁM) was greatly enhanced by replacing phenoxyꢀether part in I with corresponding
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8
9
heterocycle moiety. It was interesting to investigate the difference of the binding
among Ic, 10a, 17h and 21e. A newly hydrogen bond between Leu264 and
heterocycles moiety of 10a, 17h or 21e can be seen respectively, comparing with Ic.
This observation showed that this heterocycle moiety not only preserved the πꢀπ
interaction but also formed newly hydrogen bond with amino acid residues at the
binding site of PDHcꢀE1. The significant improvement of inhibition against E. coli
PDHcꢀE1 relative to Ic is likely due to such interactions of 10a, 17h or 21e targeting
PDHcꢀE1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to verify our docking prediction for the interactions of these novel
inhibitors with E. coli PDHcꢀE1, 17h and 21e with higher inhibition were further
studied by siteꢀdirected mutagenesis and enzymatic assays. The IC50 values of 17h or
2
1e against the mutants M194A (23.89 ꢁM or 25.68 ꢁM), E571A (16.59 ꢁM or 8.94
M), V192A (48.78 ꢁM or 28.91 ꢁM), H142A (9.2 ꢁM or 27.1 ꢁM), and L264A
ꢁ
(29.89 ꢁM or 15.48 ꢁM) were much higher than the IC50 values against wildꢀtype
PDHcꢀE1 (3.72 ꢁM or 2.12 ꢁM), respectively (Figure 6). The results showed that the
corresponding residues, Met194, E571, Val192, H142, and Leu264 played an
important role in the binding of 17h or 21e with E. coil PDHcꢀE1. Above results
showed that the prediction of molecular docking was in accordance with the
experimental results of siteꢀdirected mutagenesis.
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