In Silico Discovery and Validation of Amide Based Small Molecule Targeting the Enzymatic Site of Shiga Toxin

Article abstract of DOI:10.1021/acs.jmedchem.6b01517

Shiga toxin (Stx), a category B biothreat agent, is a ribosome inactivating protein and toxic to human and animals. Here, we designed and synthesized small molecules that block the active site of the Stx A subunit. On the basis of binding energy, 20 molec

Full text of DOI:10.1021/acs.jmedchem.6b01517

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Article
In Silico Discovery and Validation of Amide Based Small
Molecule Targeting the Enzymatic Site of Shiga Toxin
Vinita Chauhan, Dilip Chaudhary, Uma Pathak, Nandita Saxena, and Ram Kumar Dhaked
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01517 • Publication Date (Web): 16 Nov 2016
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In Silico Discovery and Validation of Amide Based Small Molecule Targeting the
Enzymatic Site of Shiga Toxin
Vinita Chauhana, Dilip Chaudharya, Uma Pathakb, Nandita Saxenac and Ram Kumar
Dhakeda∗
Biotechnology Divisiona, Synthetic Chemistry Divisionb, Pharmacology and Toxicology
Divisionc, Defence Research and Development Establishment, Gwaliorꢀ474002, India
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Abstract
Shiga toxin (Stx), a category B biothreat agent is ribosome inactivating protein and toxic to
human and animals. Here, we designed and synthesized small molecules that block active site
of Stx A subunit. Based on binding energy twenty molecules were selected for synthesis and
evaluation. These molecules were primarily screened using fluorescenceꢀbased thermal shift
assay and in vitro in Vero cells. Among 32 molecules (including 12 reported), six molecules
offered protection with IC50 value of 2.60 to 23.90 µM. 4ꢀNitroꢀNꢀ[2ꢀ(2ꢀphenylsulfanylꢀ
ethylamino)ꢀethyl]ꢀbenzamide hydrochloride is the most potent inhibitor with IC50 at 7.96 ꢁM
and selectivity index of 22.23 and is better than any known small molecule inhibitor of Stx.
Preincubation with Stx offered full protection against Shiga toxin in mice. Surface plasmon
resonance assay further confirmed that these molecules bind specifically to Stx A subunit.
Further optimization is continued to identify potential candidate which will be in vivo
effective.
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Introduction
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Shiga toxin (Stx) and Shigaꢀlike toxins (Stxꢀ1 and Stxꢀ2) are structurally and functionally
related toxins which are produced by Shigella dysenteriae type I and few strains of
Escherichia coli respectively.1,2 These toxins are listed as category B biological warfare
agents by Centers for Disease Control and Prevention (CDC) and are responsible for life
threatening bloody diarrhoea, hemorrhagic colitis, and fatal hemorrhagic uremic syndrome
(HUS). So far, no effective therapy is available for Stx intoxication. Antimicrobial treatment
is associated with increased risk of toxicity due to release of additional toxins after bacterial
cell lysis.3 Hence, treatment mainly relies on supportive therapy.
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Stx belongs to the large family of ribosome inactivating proteins (RIPs) that irreversibly
inactivate ribosome by removing specific adenine from the 28S rRNA resulting in inhibition
of protein biosynthesis. Stx is constituted by nonꢀcovalent association of single A subunit
with pentameric B subunit. A subunit is responsible for its Nꢀglycosidase activity that blocks
protein synthesis. While B subunit mediates retrograde trafficking of Stx which leads to
productive infection.4, 5
A number of approaches are being used to develop treatment against Stx toxicity.
Small molecules are of particular interest due to ease of access of a variety of desired
compounds, convenient handling, reasonably stable and are suited for oral administration.
Since retrograde transport is absolutely necessary for Stx toxicity, blocking of the toxin
transport appears promising therapeutics. Recently small molecule inhibitors Retro 1 and 2
are reported which block retrograde trafficking of Stx from early endosome to transꢀGolgi
network thus protecting cells from the Stx cytotoxicity.6 These compounds are also shown to
provide protection in mice against lethal dose of Stx and ricin which is a phytotoxin, another
member of RIP family.6ꢀ8 Noel and coꢀworkers reported further modifications of retro 2
compound by cyclization with improved protection efficacy against Stx toxicity.9 Developing
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small molecules that selectively target enzymatic activity of toxin can be safe and alternative
option for the treatment.8 NꢀGlycosidase activity of A subunit cleaves a highly conserved
adenine nucleotide within the sarcinꢀricin loop of eukaryotic 28S rRNA and leads to
ribosome arrest and inhibit protein synthesis.10 Since Nꢀglycosidase activity removes
adenines from 28S rRNA, addition of adenine protects ribosomes from inactivation by RIPs.
Several adenine derivatives and analogues have been shown to inhibit toxicity by in vitro
translation assay.11
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In the present study we have identified and synthesized small molecules targeting Nꢀ
glycosidase activity of Stx A subunit. In addition to these molecules we have also studied
twelve previously reported purine derivatives and their analogues as an inhibitor of Stx
activity of RNA and DNA depurination.11 These small molecules were initially screened
using fluorescent based thermal shift assay (FTS) and in in vitro assay using Vero cells.
Compounds which were found to be promising in initial screening were further evaluated for
their efficacy against Stx in Balb/c mice. Interaction of small molecule inhibitor with Stx is
further validated by surface plasmon resonance (SPR). Interestingly, molecules which
offered protection were all amide derivatives. Further optimization of these compounds may
uncover drug lead against Stx toxin.
Results
In silico Screening of Small Molecule Inhibitors
In an effort to identify potential inhibitors against Stx, 300 molecules were mined from
NCBI, ChemBridge and ZINC databases. These molecules were subjected to in silico studies
and docked on the active site of A chain of Stx using AutoDock 4.2. Scoring was done on the
basis of minimum binding energy (kcal/mol) of proteinꢀligand complex formation. Of them
most potent, twenty molecules were prioritized for in vitro screening. The binding energy
varied from ꢀ10.06 to ꢀ4.59 kcal/mol and Ki values from 3.21 to 439.63 µM (Table1). Among
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these; 1, 2, 3, 12, 16 and 18 are novel molecules and rest of the molecules are the reported,
however to the best of our knowledge they have never been studied against Shiga toxin.
These molecules were found to interact with the active site residues of the Stx A subunit
which indicated their probability of efficient binding thus inhibition of the toxin. The docked
models of top scoring compounds were visualized using UCSF Chimera12
(http://www.cgl.ucsf.edu/chimera), to observe the interactions between the lead compounds
and target protein (Figure S1). In order to perform the comparative analysis, previously
reported twelve small molecule Stx inhibitors (Rꢀ1 to Rꢀ12) were also included in the study
and the docking was done for these compounds using Autodock 4.2 (Table S1).
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Table 1: Chemical structure of the synthesized compounds tested as inhibitors of the RNAꢀNꢀglycosidase activity of Stx. Binding energy of the
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molecules were (compounds 1ꢀ20) considered for in silico screening using AutoDock 4.2. Table is showing name, structure, binding energy
(BE) and inhibitory constant (Ki) of the compounds.
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Binding Energy
(kcal/mole)
Ki
Compound
Number
(µM)
Structure
Name
Nꢀ(2ꢀ(phenylthio)ethyl)ꢀ2ꢀ(pyrrolidinꢀ1ꢀ
N
1
ꢀ5.64
73.78
H
yl) ethanamine hydrochloride
N
S
S
O,Oꢀdiethyl(2ꢀ{[2ꢀ
H
N
O
P
2
3
(butylsulfanyl)ethyl]amino}ethyl)phosph
oramidoꢀthioate hydrochloride
ꢀ5.04
ꢀ5.94
53.78
42.46
N
H
S
O
4ꢀNitroꢀNꢀ[2ꢀ(2ꢀphenylsulfanylꢀ
ethylamino)ꢀethyl]ꢀbenzamide
hydrochloride
S
O
O
HN
N+
ꢀ
NH
O
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O
H
N
Nꢀ(2ꢀAcetylꢀphenyl)ꢀ3ꢀphenylꢀ
acrylamide
O
4
ꢀ7.06
6.69
9
(Z)
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O
(E)
Nꢀ(2ꢀHydroxyꢀphenyl)ꢀ3ꢀphenylꢀ
acrylamide
NH
5
6
ꢀ6.45
ꢀ6.05
18.68
37.05
OH
(Z)
OH
Nꢀ(2ꢀHydroxyꢀethyl)ꢀ3ꢀphenylꢀ
acrylamide
O
N
H
O
O
(E)
3ꢀPhenylꢀacrylic acid 4ꢀoxoꢀ2ꢀphenylꢀ4
hꢀchromenꢀ3ꢀyl ester
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8
ꢀ6.64
ꢀ6.83
8.78
9.93
O
(E)
O
O
(E)ꢀ1ꢀ(2ꢀhydroxyphenyl)ꢀ3ꢀ(4ꢀ
methoxyphenyl)propꢀ2ꢀenꢀ1ꢀone
(E)
C
O
OH
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(E)
Cl
O
C
3ꢀ(4ꢀChloroꢀphenyl)ꢀ1ꢀ(2ꢀhydroxyꢀ
phenyl)ꢀpropenone
OH
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ꢀ6.77
ꢀ7.35
10.88
4.10
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O
(E)
C
OH
3ꢀ(2ꢀFluoroꢀphenyl)ꢀ1ꢀ(2ꢀhydroxyꢀ
phenyl)ꢀpropenone
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F
O
C
OH
S
(E)
1ꢀ(2ꢀHydroxyꢀphenyl)ꢀ3ꢀthiophenꢀ2ꢀylꢀ
propenone
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12
ꢀ7.16
ꢀ5.49
5.96
4ꢀChloroꢀNꢀ[2ꢀ(2ꢀpropylsulfanylꢀ
ethylamino)ꢀethyl]ꢀbenzamide
hydrochloride
O
S
HN
Cl
93.86
NH
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O
6
7
8
9
(Z)
13
14
2ꢀStyrylꢀ1Hꢀquinolinꢀ4ꢀone
ꢀ10.06
41.99
(E)
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N
H
O
F
(Z)
2ꢀ(2ꢀFluoroꢀphenyl)ꢀ1Hꢀquinolinꢀ4ꢀone
Nꢀ(2ꢀAcetylꢀphenyl)ꢀ2ꢀfluoroꢀbenzamide
ꢀ7.49
3.21
N
H
O
F
H
N
15
16
ꢀ4.59
ꢀ5.08
439.63
189.36
O
S
O
Nꢀ(1ꢀMethyleneꢀ3ꢀphenylꢀallyl)ꢀN'ꢀ(2ꢀ
propylsulfanylꢀethyl)ꢀethaneꢀ1,2ꢀdiamine
HN
NH(E)
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NH
2ꢀ(3ꢀMethylꢀthiophenꢀ2ꢀyl)ꢀ1Hꢀquinolinꢀ
4ꢀone
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18
19
ꢀ6.66
ꢀ 6.09
ꢀ 7.23
13.33
34.50
5.00
(Z)
S
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O
S
Nꢀ(2ꢀ{[2ꢀ(phenylsulfanyl)ethyl]
amino}ethyl) cyclohexane carboxamide
hydrochloride
O
HN
NH
O
H
N
Nꢀ(2ꢀAcetylꢀphenyl)ꢀ4ꢀnitroꢀbenzamide
Nꢀ(2ꢀAcetylꢀphenyl)ꢀ4ꢀfluoroꢀbenzamide
O
O
NH
O
20
ꢀ6.16
3.36
F
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Synthesis of Small Molecules
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Compound 1 was prepared by treating phenylꢀSꢀethylamine hydrochloride with 1ꢀ(2ꢀ
chloroethyl) pyrrolidine in the presence of triethylamine as a base. Compound 2 was prepared
by selective thioꢀphosphorylation of N1ꢀ(2ꢀbutylsulfanylethyl)ethaneꢀ1,2ꢀdiamine with
diethylchlorothiophosphate. Selective amidation of N1ꢀ(2ꢀphenylsulfanylethyl)ethanꢀ1,2ꢀ
diamine with corresponding acyl halide resulted in formation of Compounds 3 and 18.
Amides 4, 15, 19 and 20 were prepared by direct acylation of appropriate amine with acyl
halide. Similarly, selective amidation of N1ꢀ(2ꢀpropylsulfanylꢀethyl) ethanꢀ1,2ꢀdiamine with
4ꢀchlorobenzoyl chloride and cinnamoyl chloride yielded 12 and 16, respectively. Acylation
of 2ꢀaminophenol and 2ꢀaminoethanol with cinnamoyl chloride in water utilizing sodium bi
carbonate as base resulted in the formation of compounds 5 and 6, respectively. Compound 7
was prepared by esterifying 3ꢀHydroxyflavone with cinnamoyl chloride. Quinolones
compounds 13, 14 and 17 were resulted by cyclisation of the amides obtained from 2ꢀ
aminoacetophenone and corresponding acyl halides. Chalcones ie compounds 8, 9, 10 and 11
were prepared by the method used in reported literature.13, 14
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Primary Screening using FluorescenceꢀBased Thermal Shift Assay (FTS)
In present study, FTS assay was used for initial screening of synthesized molecules for their
interaction with recombinant Stx A subunit (rStx A). Thermal stability shift analysis is a fast
and powerful platform for screening of molecules that affects thermal stability of proteins
after binding interactions.15 This assay operates on the principle that each protein unfolds at
specific temperature and binding of ligands alters the thermal stability of the protein resulting
in shift in unfolding temperature. A probe (sypro orange) provides fluorescence due to
binding to hydrophobic surface resulted from unfolding of the protein. All 32 compounds
were evaluated for their interaction with rStx A subunit. Seven molecules are found to bind
o
with greater affinity and altered the stability of protein, resulting >2 C change in transition
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temperature (ꢀTm). Among these seven compounds, maximum shift in ꢀTm was recorded
with compound 15 (7.25°C) followed by 4 (4.61 °C), Rꢀ7 (4.45 °C), 3 (3.87 °C), Rꢀ8 (3.75
°C), 16 (2.65 °C) and Rꢀ2 (2.32 °C) (Figure 1 and Table S1).
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8
7.25
6
4.61
3.87
4
2
2.65
0
-2
-4
-6
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Compound Number
Figure 1: Fluorescence based thermal shift assay result for interaction between small
molecules and Stx. The Tm depicts the difference between in the Tm of Stx mixed with
ꢀ
ligand and the Tm of Stx only reactions Values are average of three replicates. Small
molecules showing various degrees of thermal shift ranging from ꢀ4.16 °C to 7.25 °C.
Stx Induced Cytotoxicity in a Dose and Time Dependent Manner in Vero Cells
To examine the cytotoxic effect and 50% cytotoxic concentration (CC50) of Stx in cells, the
Vero cells were treated with different concentration of Stx ranging from 0 to 120 ng/ml for 24
h, and the viability was determined by3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyltetrazolium
bromide (MTT) and neutral red (NR) dye uptake assay. From 20 ng/ml, Stx caused a
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significant dose dependent decrease in viability with increase in concentration at 24h.
Concentration that is 50% cytotoxic (CC50) was found to be 100 ng/ml at 24 h (Figure 2A).
This CC50 value of 100 ng/ml was used in further experiments.
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Effect of Compound on Cells and Stx Induced Toxicity
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All 32 small molecules were evaluated for maximum tolerated dose (MTD) in Vero cells. For
determination of MTD, cells were treated with varying concentration of compounds for 24 h
and then cell viability was measured by NR assay. MTD values of synthesized compounds
varied from 5.00±1.23 µM to 123.00±2.45 µM and for reported compounds 9.30±1.53 µM to
250.00±1.75 µM. (Table 2A and 2B). Further, to test the efficacy of the small molecules as
an inhibitor of Stx by NR assay, cells were preꢀtreated with compounds for 1h followed by
Stx exposure. Among the 32 compounds evaluated, four synthesized compounds (3, 4, 15 and
16) and two reported compounds (Rꢀ7 and Rꢀ8) showed 75ꢀ 95% inhibition of Stx induced
toxicity (Figure 2 B and C). Efficacy of these six compounds was further validated by MTT
and lactate dehydrogenase (LDH) release assay. As shown in figure 2D these small molecules
were found to protect cells from Stx toxicity by MTT assay. Similarly, these compounds also
showed significant inhibition of Stx induced LDH release and protected cell membrane
integrity (Figure 2E). Other compounds showed no significant reversal of Stx induced
toxicity in Vero cells. Those compounds which offered protection was also tested for their
dose depended potential to inhibit Stx toxicity. In Figure S2 (supporting information)
compound 3 has been shown to inhibit Stx toxicity in dose dependent manner with highest
inhibition at approximately 12 µM. The doses of compounds (3, 4, 15, 16, Rꢀ7 and Rꢀ8)
inhibiting 50% toxicity of Stx (IC50) were determined in cells treated with different
concentrations of compounds by NR assay (Table 2). These six compounds were also tested
for their 50% cytotoxic concentration (CC50) using NR assay and results are mentioned in
table 2. Based on the CC50 and IC50 values, the selectivity index (SI=CC50/IC50) of each
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compound was determined which reflects the quantity of compound that is active against the
toxin but is not toxic towards the host cell. SI of compounds which offered protection were
22.23, 11.29, 15.50 and 15.00 for compounds 3, 4, 15 and 6, respectively. Although,
compounds 3, 4, 15 and 16 significantly inhibited the Stx toxin induced cell death, but
compound 3 was found to be most potent with SI value of 22.23 (Table 2A). Among the
purine derivatives included in the study, guanine (Rꢀ7) showed maximum 88% inhibition and
IC50 value of 11.50 µM and 76% inhibition was observed with allopurinol (Rꢀ8) with an IC50
value of 23.90 µM. The SI values were found to be 11.80 and 17.90 for Rꢀ7 and Rꢀ8,
respectively (Table 2B).
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Figure: 2
(A)
100
∗
80
∗
∗
∗
∗
∗
∗
60
40
20
0
∗
∗
∗
∗
∗
MTT assay
NR assay
0
20
40
60
80
100
120
Stx (ng/ml)
(B)
120
#
#
100
80
60
40
20
0
#
#
#
C Stx 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
13
Compound Number
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Compound code
(D)
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#
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Stx
3
4
15
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R-7
R-8
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Stx
3
4
15
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R-7
R-8
Compound Number
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Figure 2: Stxꢀinduced cytotoxicity and evaluation of small molecule inhibitors for in vitro
protection in Vero cells. (A), Stxꢀinduced toxicity: Cytotoxic effect of Stx in Vero cells was
determined by MTT and NR assays. For determination of 50% cytotoxic concentration
(CC50), the cells were seeded on to a 96 well plate and treated with varying concentration of
Stx for 24 h. The CC50 was calculated as 100 ng/ml. Data are the mean of three independent
experiments each in triplicate with standard error *p<0.05 compared with untreated cells. (B),
Preꢀtreatment of compounds followed by Stx (100 ng/ml) exposure for 24 h. Cells were
analyzed by NR assay. Compounds 3, 4, 15 and 16, significantly reversed Stx induced
cytotoxicity. Data are the mean of three independent experiments each in triplicates with
standard error. #p<0.05 compare to Stx treated cell, (C), Preꢀtreatment of previously reported
compounds followed by Stx (100 ng/ml) exposure for 24 h. Cells were analyzed by NR
assay. Compounds Rꢀ7 and Rꢀ8 were found to be promising; Data are the mean of three
independent experiments each in triplicates with standard error. #p<0.05 compare to Stx
treated cell, (D), Preꢀtreatment of compounds 3, 4, 15, 16, Rꢀ7 and Rꢀ8 followed by Stx (100
ng/ml) exposure for 24 h. Cells were analyzed by MTT assay. All six small molecule
inhibitor offered protection against Stx induced toxicity. Data are the mean of three
independent experiments each in triplicates with standard error. #p<0.05 compare to Stx
treated cell, (E), Preꢀtreatment of compounds 3, 4, 15, 16, Rꢀ7 and Rꢀ8 followed by Stx (100
ng/ml) exposure for 24 h. Cells were analyzed by lactate dehydrogenase (LDH) release assay.
All six compounds could significantly block Stx induced LDH release. Data are the mean of
three independent experiments each in triplicates with standard error. #p<0.05 compare to Stx
treated cell. Untreated cells (C), only toxin treated (Stx), Pretreatment of compounds for 1 h
(Compounds #1ꢀ20 and Rꢀ1 to Rꢀ12) followed by Stx.
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Table 2: Determination of maximum tolerated dose (MTD), 50% cytotoxic concentration
5
6
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8
(CC50), inhibitory concentration (IC50) and selectivity index (SI) of the small molecules in
Vero cells; (A) synthesized compounds; (B) reported compounds.
Table 2A
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MTD
CC50
IC50
Compound Code
SI
(ꢁM)
(ꢁM)
(ꢁM)
1
2
63.30±1.53
32.30±1.72
15.00±1.01
6.96±1.00
32.00±2.65
31.60±1.53
64.00±2.00
7.35±0.58
6.00±2.05
5.67±1.64
17.00±.3.06
123.60±2.45
9.33±1.53
5.00±1.23
37.00± 1.00
38.00±2.36
5.33±1.00
38.00±1.45
17.33±1.53
10.00± 1.23
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
177.00±7.10
7.96±1.90
22.23
4
29.71±5.40
02.63±0.53
11.29
5
ꢀ
ꢀ
ꢀ
6
ꢀ
ꢀ
ꢀ
7
ꢀ
ꢀ
ꢀ
8
ꢀ
ꢀ
ꢀ
9
ꢀ
ꢀ
ꢀ
10
11
12
13
14
15
16
17
18
19
20
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
120.00±3.24
7.70±1.10
15.50
123.00±4.23
8.20±0.50
15.00
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
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Table 2B
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MTD
(ꢁM)
9.30±2.30
CC50
(ꢁM)
ꢀ
IC50
(ꢁM)
ꢀ
Compound code
SI
ꢀ
Rꢀ1
Rꢀ2
Rꢀ3
Rꢀ4
Rꢀ5
Rꢀ6
Rꢀ7
Rꢀ8
Rꢀ9
Rꢀ10
Rꢀ11
Rꢀ12
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125.00±3.24
18.70±5.60
125.00±1.50
9.30 ±2.80
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.30±1.53
ꢀ
ꢀ
ꢀ
125.00±1.87
250.00 ±1.75
250.00±3.60
18.70±4.20
125.00±3.20
75.00±3.45
136.50±2.50
11.50±0.90
11.80
428.00±5.60
23.90±2.00
17.90
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Binding Studies of Small Molecule Inhibitors with Stx A
To quantitatively assess the binding affinity of compounds 3, 4, 15 and 16 with Stx we
performed SPR experiments using immobilized rStxA on GLH sensor chips at ~6000 RU
(resonance units) as described in experimental section. As shown in figure 3, compound 3
binds to rStxA in a concentration dependent manner with high affinity and yielding the
equilibrium dissociation constant (KD) of 7.16X10ꢀ7 M with a fast on rate (
k
a=9.46X103
1/Ms) and fast off rate (
k
d=6.77X10ꢀ21/s). Other compounds 4, 15 and 16 also interacted to
StxA but comparatively with lower affinities yielding KDs of 3.09X10ꢀ6, 9.68X10ꢀ2 and
8.67X10ꢀ2 M, respectively (figure S3).
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Figure 3: Surface plasmon resonance experiments to analyze the binding curves and fitted
results of compound 3 to immobilized rStxA on GLH sensor chip. The concentrations of
compound used were ranging from 25.00 to 1.625 µM. PBS with tweenꢀ20 was used as a
control. The data were fitted to the simplest Langmuir 1∶1 interaction model. The inset is
showing the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant
binding (KD) constants.
In vivo Evaluation of Compound 3, 4, 15, 16 and Rꢀ7, Rꢀ8 in Mouse Model
To assess whether our finding is restricted to cultured cell lines, we analyzed the efficacy of
compounds in mice against supra lethal dose of Stx (5x LD50). In Stx treated group, all mice
died between 12 to 24 h with symptoms of shigellosis. The groups in which mice were
exposed to Stx with pre or post treatment of compounds resulted in the extension of survival
time where mice death was recorded in 24 to 48 h. Interestingly, in the group where
compounds were preꢀincubated with 5x LD50 of Stx and then administered, all the animals
survived and no animal showed any symptomatic effect of toxin (figure 4). This finding
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suggests neutralizing potential of these compounds against Shiga toxin toxicity. Surprisingly,
Rꢀ7 and Rꢀ8 did not offer any protection even when they administered after preꢀincubating
with Stx. In vehicle control and treatment with compound alone groups did not show any sign
of toxicity (data not shown).
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Figure: 4
Figure 4: In vivo efficacy studies of small molecules. Balb/c mice were administered
intraperitoneally with 5x LD50 of Stx. Compounds were given either 1h before or 1 h after
treatment of Stx. In another group Stx and compounds were premixed and then administered
to mice. For all treatment 2 mM compounds was used.
Discussion
Stx is a category B biothreat agent, therapeutic agents that inhibit Stx toxicity are urgently
required to prevent the harmful consequences of Stx that are not addressed with current
antibioticꢀbased treatments. Various novel approaches have been attempted to develop
inhibitors of various stages of Stx intoxication including cell uptake, trafficking and release to
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the cytoplasm.6,7,9,16ꢀ20 The small molecules identified using high throughput screening that
inhibit endogenous trafficking of toxin may affect other endogenous cargo proteins and can
lead to other undesirable effects. Hence, only those molecules that directly interfere with
active site and inhibits its Nꢀglycosidase activity may be helpful.
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Structure based small molecule inhibitors is an attractive approach for the treatment of Stx
intoxication. In this study, we utilized in silico screening to identify Stx inhibitors selectively
targeting NꢀGlycosidase activity of A subunit of Stx. Initial molecular screening of several
small molecules i.e. amides, chalcones, esters and quinolones, led us to identify the best
predicted inhibitors against shiga toxin. Finally, based on binding energy and Ki, we
identified 20 molecules for synthesis and screening. Twelve reported purine derivatives were
also included in the study.11 These molecules act as an uncompetitive inhibitor of the RNAꢀ
Nꢀglycosidase reaction and protect inactivation of ribosome by Stx.
Initial screening of these compounds was done using FTS assay and in vitro assay. FTS
assay is an inexpensive method for screening of small molecule inhibitors that rely on change
in thermal stability of the proteins after binding of the ligands.15 Among 32 compounds
evaluated, seven compounds were found to alter the stability of rStxA, where shift in
o
transition temperature (
ꢀ
Tm) was recorded >2 C. These 32 compounds were also evaluated
in cell based assay against Shiga toxin, out of those six compounds offered protection, four of
them were new compounds (3, 4, 15 and 16) and two were reported inhibitors (Rꢀ7 and Rꢀ8).
Interestingly, all the newly identified compounds were amides, suggesting amide derivatives
may have potential to inhibit catalytic activity of Stx. We observed a good agreement
between our initial screening methods, FTS and cell based assay. The compounds which were
showing maximum thermal shift were consistent with the result that these small molecules
owe the most potent in vitro inhibition activity. The only exception was observed with Rꢀ2
that was not able to protect cells after Stx challenge (table 2A & B).
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Selectivity index (SI) value defines the compound’s ability to preferentially produce a
particular effect and is important parameter for analysing protective effect of compounds.
Among all the compounds studied, compound 3 was found to be most promising in terms of
protection against Stx with the highest SI value of 22.23. The magnitude of inhibition was
also higher than those of earlier reported small molecule inhibitors including retro 1 and 2
where the IC50 was reported to be at 25.00 ꢁM in cell culture.19 Whereas in present study,
IC50 of lead compounds was varied from 2.63±0.53 to 8.20±0.50 ꢁM. Lower IC50 value in
cell based protection assay reflects the higher efficacy of inhibitors, suggesting the greater
therapeutic potential of present leads.
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Evaluation of molecules in Vero cells produced interesting results. Compounds having amide
functionality were found very effective, while chalcones, quinolones and ester of 3ꢀ
hydroxyflavone did not provide any protection. Compounds 1, 3 and 18 were relatively
similar except the terminal amide moiety. The modification of terminal amine functionality to
pyrrolidine (compound 1) was not found effective. In 3 and 18 the terminal amine was
acylated with 4ꢀnitrobenzoylchloride (aromatic moiety) and cyclohexane carbonyl chloride
(alicyclic moiety), respectively to generate the amide group. Amidation with aromatic moiety
(compound 3) was found much more effective than alicyclic moiety (compound 18).
Compound 3 contained two aromatic rings with one amine and one amide functionality was
the most effective inhibitor in cell culture studies. Among the cinnamides, 4, 5, 6 and 16; the
most effective compound was 16 followed by 4. Cyclisation of amides derived from 2ꢀamino
acetophenonequinolone, 14 and 17 were ineffective in cytoprotection and at the same time
compound toxicity was also increased. Placing fluoro group at ortho position (compound 15),
compared to para position in aromatic ring (compound 20) proved to be more useful, as
compound 15 was impressively effective while other did not provide any protection.
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Evaluation of reported compound in Vero cells also revealed interesting results. Guanine (Rꢀ
7) and allopurinol (Rꢀ8) are reported the least efficient inhibitors of RNAꢀN glycosidase
activity by in vitro protein synthesis assay but same showed highest efficacy in Vero cells
against Stx in our study.11 Probably cell based assay used in present study is closer to
biological system than in vitro protein synthesis inhibition assay. It could also be possible
that both compounds may exhibit some additional effect like interference of toxin binding to
its receptor, trafficking or processing in the ER. 4ꢀAminopyrazolo[3,4ꢀd]pyrimidine (4ꢀ
APP), which was reported to be most efficient small molecule inhibitor could not be
evaluated due to the nonꢀavailability of the compound with us.11 Guanine was also showing
good inhibition against enzymatic activity of ricin, substantiate the hope for finding common
inhibitor working against all RIP.17
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To investigate the possibility of nonꢀspecific or multiꢀtargets binding of these leads, pan
assay interference compounds (PAINS) filter was applied. None of the lead molecules
(compounds 3, 4, 15 and 16) were recognized as potential PAINS. Moreover, screening
system used in in vitro evaluation is based on Vero cell viability for the estimation of
potential inhibitor unlike other HTS methods where above mentioned interference are major
hurdle.21 Compound 5 having the substructure included in PAINS was also evaluated in cell
based assay and showed no inhibitory activity, this further strengthen that compounds 3, 4,
15 and 16 are true positive hits.
SPR is a highly sensitive label free technique to measure binding and the affinities of
potential inhibitors to ligand. It provides information about kinetics of interaction and the
halfꢀlife of the complex. These biosensors can be used for secondary screening to confirm
whether the hit is real and can also provide information about their dynamic interaction in
realꢀtime. SPR experimentation showed that the amide based small molecules were
interacting with rStxA in concentration dependent manner with high affinities and
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equilibrium dissociation constants (KD) varied from 7.16X10ꢀ7 to 8.67X10ꢀ2 M. There was
considerable correlation in SI values obtained from cytoprotection assay and the KDs of
interactions between rStxA subunit and amide based compounds. On the basis of both values
compound 3 was found to be most effective. The only exception was compound 4 which
provided least SI value but interacted with higher KD than compound 15 and 16. As shown in
figure 5, when compound 3 is docked in the active site pocket of Stx, the amide group of
compound forms a hydrogen bond with Ser112 and is interacting with active site of toxin i.e.
Tyr77, Val78, Tyr114, Glu167, Ala168, Trp203 and Arg170, thus contributes to the
specificity and potency of this inhibitor. The amide of compound 4 formed a hydrogen bond
with Arg170 that is reported to involve in substrate binding.17 In docked conformations
compound 15 was found to interact with Arg170 and Tyr114, whereas compound 16 was
seen interacting with Ser112 and Val78, which are reported as active site residues of Stx.
Our findings from in vivo assay revealed that all 4 amide compounds are highly effective in
neutralizing toxin by preꢀincubating and protecting mice against Stx toxicity. There were no
symptoms observed of Stx toxicity in animals. Protection obtained by preꢀincubating
indicates the efficacy of lead compound as Stx inhibitor and their potential to neutralize
extracellular toxin. Significant extension in survival time was observed when the mice were
challenged with Stx followed by preꢀ and postꢀtreatment of inhibitors. However, no
protection suggests the possibility of modification of compound or pharmacokinetics inside
biological system. This may also be due to distribution, absorption or/and stability of the
compound in body system. Both the earlier reported compounds (guanine and allopurinol)
were not found effective in all three modes of treatment. Though the efficacy of leads
identified in present study needs further optimization with respect to neutralization of toxin in
preꢀ and postꢀchallenge. All these leads are amide derivatives but compound 3 was found to
be most promising and efficacious and require further study. Further these compounds are
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nonꢀoptimized hits and modification in the chemistry of compounds may further improve in
vivo efficacy.
7
8
Conclusion
9
Our findings indicate that targeting the catalytic site with small molecules represent a feasible
therapeutic approach that may allow successful reversal of Shiga toxicity. Since our
molecules are specific inhibitors of enzymatic activity of Shiga toxin hence they do not cause
any undesirable effects unlike inhibitors of retrograde pathway which may affect trafficking
of other vital endogenous cargo protein. Among all the compounds studied, 4ꢀNitroꢀNꢀ[2ꢀ(2ꢀ
phenylsulfanylꢀethylamino)ꢀethyl]ꢀbenzamide hydrochloride (compound 3) is found to be
most promising in terms of protection against Stx with the highest SI value of 22.23 and the
magnitude of inhibition is higher than those of earlier reported small molecules with the IC50
of 2.63±0.53 ꢁM. Preꢀincubation of Shiga toxin with these amides based small molecules
also protected animals. Lower IC50 values in cell based assay reflects the higher efficacy of
inhibitors, suggesting the greater therapeutic potential and their activities can be enhanced by
further lead optimization efforts which are under progress.
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Figure: 5
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Figure 5: Close view of the compounds 3, 4, 15, 16, Rꢀ7 and Rꢀ8 that offered protection, to
show the interaction and active site of Shiga toxin (PDB code 1DM0) with the docked
ligands using UCSF chimera.
5(A): Hydrogen bond between amide group of compound 3 and Ser112.
5(B): The amide group of compound 4 forming hydrogen bond with Arg170.
5(C): Compound 15 is located in pocket surrounded by active site residues Arg170 and
Tyr114.
5(D): Compound 16 is located in pocket surrounded by active site residues Ser112 and
Val78.
5(E): Rꢀ7 is located in pocket surrounded by active site residues.
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Experimental Section
1. Chemistry Methods
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Reagents of analytical grade (>98%) were obtained from commercial suppliers and used
without further purification. Column chromatography was performed on silica gel (60 ꢀ 120
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1
mesh). H, 31P and 13C NMR spectra were recorded on Bruker AVANCEII 400 MHz and
Bruker AVIII 600 MHz. Chemical shifts were expressed in parts per millions (δ) downfield
from the internal standard tetramethylsilane and were reported as s (singlet), d (doublet), t
(triplet), q (quartet) and m (multiplet). Melting points were measured by scientificꢀMPꢀDS
apparatus. Mass spectra were obtained from Agilentꢀ5975C GCꢀMS and elemental analysis
was performed on Elementar vario MICROꢀcube CHNS analyser. Purity was checked by
elemental analysis and were >98% for all the compounds synthesized. The reported
compounds (Rꢀcompounds) were purchased from SigmaꢀAldrich (St Louis, MO) and were
>99% pure except Rꢀ3, Rꢀ5 and Rꢀ9 where the purity was ~98%.
2. Synthesis of Compounds
Nꢀ(2ꢀ(phenylthio)ethyl)ꢀ2ꢀ(pyrrolidinꢀ1ꢀyl) ethanamine hydrochloride (1). PhenylꢀSꢀ
ethylamine hydrochloride 10 mmol (1.89 g) and 1ꢀ(2ꢀChloroꢀethyl)ꢀpyrrolidine 2.0 mmol
(2.60 g) was dissolved in 10 ml of ethanol. The contents were heated to reflux. To this,
triethylamine 5 mmol (7.50 ml) in 30 ml of ethanol was added dropꢀwise over a period of 3ꢀ4
h. After the addition is over reaction mixture was heated till the reaction was complete, which
was monitored by GCꢀMS and TLC (mobile phase CH3OH:CHCl3:NH3OH; 3:2:1). On
evaporation of ethanol, solid residue was obtained, which was dissolved in chloroform and
washed with sodium carbonate. Evaporation of chloroform resulted in an oily mass. The oil
was dissolved in acetone and converted into its hydrochloride salt with dry HCl. The
hydrochloride salt of Nꢀalkyl derivative was obtained as solid precipitate which was purified
by recrystallization from acetoneꢀmethanol mixture.
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White solid; mpꢀ 175ꢀ176 °C; H NMR (400 MHz, CD3OD): δ=7.42ꢀ7.40 (m, 2H), 7.33 (t,
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2H, J = 8.0 Hz), 7.28 (d, 1H, J = 6.8 Hz), 3.51ꢀ3.47 (m, 2H), 3.40ꢀ3.36 (m, 3H), 3.22 (s, 5H),
1.99 (br s, 4H); 13C NMR (150.9 MHz, CD3OD) δ 135.02, 131.58, 130.66, 128.51, 55.70,
49.57, 44.45, 30.55, 24.15, 0.11.MS (EI): m/z 55, 70, 84, 98, 117, 155. Anal. calcd for
C14H22N2S: C, 67.15; H, 8.86; N, 11.19; S, 12.81. Found: C, 67.17; H, 8.84; N, 11.17; S,
12.85.
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O,Oꢀdiethyl(2ꢀ{[2ꢀ(butylsulfanyl)ethyl]amino}ethyl)phosphoramidoꢀthioate
hydrochloride (2). A two neck flask was charged with dry dichloromethane (DCM) and N1ꢀ
(2ꢀ(butylthio) ethyl) ethaneꢀ1, 2ꢀdiamine dihydrochloride23, 10 mmol (2.12 g). The mixture
was stirred and then triethylamine was added very slowly to reaction mass. Reaction mixture
was stirred further for 5ꢀ10 min. This was followed by drop wise addition of diethyl
chlorothiophosphate dissolved in dichloromethane at 20ꢀ25 ºC. On complete addition of
diethyl chlorothiophosphate 12 mmol (2.2 ml), the reaction mixture was stirred for additional
1ꢀ2 h. The reaction was monitored by GCꢀMS. After completion of the reaction, organic layer
of DCM was washed with NaHCO3, and DCM layer was dried over anhydrous Na2SO4. The
solvent (DCM) was evaporated and an oily residue was obtained. The residue was dissolved
in acetone and acidified by dry HCl gas. The acidic acetone solution was evaporated to a half
volume by purging nitrogen. Then diethyl ether was added. White precipitate was appeared
which was filtered and washed with diethyl ether and dried under vacuum. Compound was
recrystallized by acetoneꢀether solvent system.
1
White shiny crystal; mpꢀ 99ꢀ101 °C; H NMR (400 MHz, CD3OD): δ 5.70ꢀ5.63 (m, 4H),
5.36ꢀ5.31 (m, 2H), 5.02 (d, 4H, J = 5.2 Hz), 4.78 (d, 2H, J = 5.2 Hz), 3.99 (t, 2H, J = 7.2 Hz),
2.97ꢀ2.76 (m, 2H), 2.76ꢀ2.67 (m, 6H), 2.29 (t, 3H, J = 7.2 Hz). EIMS: m/z 117, 170, 196,
225, 226; 13C NMR (150.9 MHz, CD3OD) δ 64.57, 48.25, 39.03, 32.93, 32.63, 28.68, 23.17,
16.56, 14.25, 0.24; MS (EI): m/z 61, 73, 97, 117, 135, 153, 170, 196, 225, 251. Anal. calcd
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for
C12H29N2O2PS2: C, 43.88; H, 8.90; N, 8.53; S, 19.52. Found: C, 43.85; H, 8.95; N, 8.51;
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S, 19.56.
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4ꢀNitroꢀNꢀ[2ꢀ(2ꢀphenylsulfanylꢀethylamino)ꢀethyl]ꢀbenzamide hydrochloride (3). N1ꢀ(2ꢀ
Phenylsulfanylꢀethyl)ꢀethaneꢀ1,2 diamine dihydrochloride 1 mmol (0.269 g) was taken round
bottom flask and to this 10 ml dry DCM was added. To this mixture triethylamine 2 mmol
was added very slowly. The content was stirred till the clear solution was formed. After that
1.1 mmol (0.204 g) 4ꢀ nitro ꢀbenzoyl chloride in 10 ml of DCM was added slowly (in 2 h) at
20ꢀ25 °C. Reaction was monitored by GCꢀMS and TLC (mobile phase CH3OH:CHCl3:NH3;
10:10:1). After completion of reaction, reaction mixture was washed with NaHCO3 solution
and then water. DCM layer was collected and dried over anhydrous sodium sulphate. On
evaporation of organic layer an oily residue was obtained. The residue was dissolved in
acetone and dry HCl gas was passed. Hydrochloride salts of amide derivatives were appeared
as white precipitate. The product was further purified by recrystallization from ethanolꢀ
acetone.
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White solid; mpꢀ170ꢀ172 °C; 1H NMR (400 MHz, CDCl3): δ= 8.28–8.22 (m, 2H), 8.07–8.05
(d, 1H, J = 8.4 Hz), 7.98ꢀ7.96 (d, 1H, J = 8.4 Hz), 7.52ꢀ7.11 (m, 5H), 3.91ꢀ3.80 (m, 3H),
3.50ꢀ3.47 (m, 1H), 3.34ꢀ3.28 (m, 1H), 3.15 (s, 1H), 1.55ꢀ1.50 (m, 1H); 13C NMR (150.9
MHz, CDCl3) δ 165.39, 149.53, 140.13, 129.60, 129.10, 128.26, 126.49, 123.75, 47.28,
39.48, 34.35, 29.71; MS(EI): m/z 56, 76, 109, 137, 150, 193, 222. Anal. calcd for
C17H19N3O3S: C, 59.11; H, 5.54; N, 12.17; S, 9.28. Found: C, 59.15; H, 5.55; N, 12.13; S,
9.25.
Nꢀ(2ꢀAcetylꢀphenyl)ꢀ3ꢀphenylꢀacrylamide (4). 2ꢀaminoacetophenone 10 mmol (1.3 ml) was
taken in round bottom flask and to this 20 ml dry DCM was added. Triethylamine 2 mmol (3
ml) was added slowly. The content was stirred till the clear solution was formed. After that
the cinnamoyl chloride 11 mmol (1.1 g) in 30 ml of DCM was added slowly (in 2 h) at 20ꢀ25
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