In silico and bio assay of juvenile hormone analogs as an insect growth regulator against Galleria mellonella (wax moth)-Part i

Article abstract of DOI:10.1080/07391102.2015.1056549

Juvenile hormone (JH) analogs are nowadays in use to control harmful pests. In order to develop new bioactive molecules as potential pesticides, we have incorporated different active structural features like sulfonamide, aromatic rings, amide group, and a

Full text of DOI:10.1080/07391102.2015.1056549

Journal of Biomolecular Structure and Dynamics
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In silico and bio assay of juvenile hormone analogs
as an insect growth regulator against Galleria
mellonella (wax moth) – Part I
Priyanka Sharma, Sunil Thakur & Pamita Awasthi
To cite this article: Priyanka Sharma, Sunil Thakur & Pamita Awasthi (2016): In silico and
bio assay of juvenile hormone analogs as an insect growth regulator against Galleria
mellonella (wax moth) – Part I, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2015.1056549
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Date: 13 April 2016, At: 00:28

Journal of Biomolecular Structure and Dynamics, 2016
http://dx.doi.org/10.1080/07391102.2015.1056549
In silico and bio assay of juvenile hormone analogs as an insect growth regulator against
Galleria mellonella (wax moth) – Part I
Priyanka Sharmaa, Sunil Thakurb and Pamita Awasthia*
b
aDepartment of Chemistry, National Institute of Technology, Hamirpur, HP 177005, India; Institute of Environmental Science and
Biotechnology, Hamirpur, HP 177001, India
Communicated by Ramaswamy H. Sharma
(Received 21 March 2015; accepted 26 May 2015)
Juvenile hormone (JH) analogs are nowadays in use to control harmful pests. In order to develop new bioactive molecules
as potential pesticides, we have incorporated different active structural features like sulfonamide, aromatic rings, amide
group, and amino acid moiety to the base structure. We have screened a series of designed novel JH analogs against JH
receptor protein (jhbpGm-2RCK) of Galleria mellonella in comparison to commercial insect growth regulators (IGRs) –
Pyriproxyfen (T1) and Fenoxycarb (T2). All analogs exhibit the binding energy profile comparable to commercial IGRs.
Based upon these results, a series of sulfonamide-based JHAs (T3–T8) as IGRs have been synthesized and characterized.
Further, the efficacy of synthesized analogs (T3–T8) and commercial IGRs (Pyriproxyfen and Fenoxycarb) has been
assessed against fourth instars larvae of G. mellonella under the laboratory conditions. LC50 values of all the analogs
(T1–T8) against the fourth instars larvae were 9.99, 10.12, 24.76, 30.73, 38.45, 34.15, 34.14, 19.48 ppm and the LC90
153.27, 131.69, 112.15, 191.46, 427.02, 167.13, 217.10, 172.00 ppm, respectively. Among these analogs, N-(1-isopropyl-
2-oxo-3-aza-3-N-ethyl-pentanyl)-p-toluene sulfonamide (T8) and N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene
sulfonamide (T7) exhibited the good pest larval mortality at different exposure periods (in hours) and different concentra-
tions (in ppm) in comparison to in use IGRs- T1 and T2. Bio assay results are supported by docking at higher concentra-
tion. The present investigation clearly exhibits that analog T8 could serve as a potential IGR in comparison to in use IGRs
(T1 and T2). The results are promising and provide new array of synthetic chemicals that may be utilized as IGRs.
Keywords: juvenile hormones (JHs); juvenile hormone analogs (JHAs); insect growth regulators (IGRs); juvenile
hormone binding proteins (JHBP); docking; Galleria mellonella; larval mortality; lethal concentration (LC)
1. Introduction
Several JH analogs (JHAs) have been proven to be
effective IGRs and useful as insecticides against
Lepidopteran pests (Hirano, Hatakoshi, & Kawada,
1998). Among these IGR’s, Pyriproxyfen shows high
inhibitory reproductive and metamorphic action against
agricultural, household, and public hygiene insect pest
(Figure 1(b)) (Amiri, Bandani, & Darvishzadeh, 2012;
Fahmy, 2012; Ghoneim, Hamadah, & Tanani, 2012;
Mojaver & Bandani, 2010). Fenoxycarb is also an
important IGR effective against flea, mosquito, and
cockroach. These JH analogs are presently in-use in inte-
grated pest management program worldwide (Bortolotti,
Porrini, Sbrenna, & Sbrenna, 2000; Liu & Chen, 2001).
The JH counters the target directly by gene expres-
sion or via second messengers (Sevala & Davey, 1993;
Wyatt & Davey, 1996). Since JH exert its action for
individual process through stage specific manner. It is
generally assumed that multiple JH receptors are found
in cytosol and nuclei. JH upon releasing from corpora
allata binds to low molecular weight – juvenile hormone
Juvenile hormones (JHs), an acyclic chain of sesquiter-
penoid, organize different processes including growth,
development, metamorphosis, and reproduction in insects
(Gilbert, Granger, & Roe, 2000; Truman & Riddiford,
1999) (Figure 1 (a)). These are also referred as “third
generation pesticides” as it is the time and amount of JH
secreted by endocrine system of insect control the life
cycle of insect from the immature larval stage to the
adult form. Hence, JH – agonist or antagonist activity –
has become the choice for insect pest control due to their
species specificity (Tunaz & Uygun, 2004).
Insect growth regulators (IGRs) are the compounds
which regulate the growth of insect and consider to play
a vital role in integrated pest management systems. Con-
ventional insecticides have some shortcomings such as
slowness of insecticidal activity, pest resistance, and
environmental concern, therefore, designing and develop-
ment of IGR to be potential target specific pesticide is
new and emerging area of research.
*Corresponding author. Email: pamita@nith.ac.in
© 2016 Informa UK Limited, trading as Taylor & Francis Group

2
P. Sharma et al.
Figure 1. (a) Natural JH; (b) synthetic IGRs (T1–T2); (c) structure of parent analog of designed series (T3–T8).
binding protein (JHBP) present in hemolymph. Complex
formation between JH and JHBP provides protection to
JH (labile) against non-specific enzymatic degradation.
Therefore, JHBP is regarded as one of the most impor-
tant protein that regulates the JH signaling (Fujimoto
et al., 2013; Hidayat & Goodman, 1994; Suzuki et al.,
2011; Touhara, Lerro, Bonning, Hammock, & Prestwich,
1993). JHBPs have been extracted from lepidopteron
insect species (M. sexta, B. mori, H. virescens, Galleria
mellonella, A. gambiae, D. melanogaster) showed a
strong sequence similarity (Gaubard, 2005). Hence,
JHBP is expected to be an excellent target for the
development of novel species specific IGRs. A better
understanding of the JHBP will provide new platform
for the structure-based ligand design.
G. mellonella and JHBP of G. mellonella is an object
of present study. It is commonly known as greater wax
moth and considered to be a harmful beehive pest in the
world. It feeds on honey bee larvae, ingest honey, pollen,
and wax. Crystal structure of JHBP of G. mellonella has
been studied deeply using X-ray crystallography tool
(Kolodziejczyk et al., 2008). Elongated structure of the
JHBP with long α helix wrapped in a highly curved anti-
parallel β sheet with two binding cavities (hydrophobic

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
3
W and E). Each cavity is created between long α4 and
set of β strands with 4.0 Ǻ × diameter. Further, experi-
mentally it has been proposed that JHBP undergoes con-
formational changes upon binding with JH. It has been
suggested that these structural changes affect target cell
membrane which is proposed to be significant for hor-
monal signaling (Kolodziejczyk et al., 2008). Numerous
analogs of JH with different functionalities have been
synthesized and studied extensively against diverse
harmful pest species. Also combination of different struc-
tural functionalities at the terpenoids and non-terpenoids
skeleton proved to have significant effect on the biologi-
cal activities of JHA (Awasthi & Mahajan, 2008;
Devillers, 2013; Frings, Thomé, & Bolm, 2012; He
et al., 2013; Kad & Kalra, 1999; Kukovinets, Zvereva,
Kasradze, Abdullina, & Tolstikov, 2008; Odinokov,
Kukovinets, Zainullin, & Tolstikov, 1992; Svobodova
et al., 2010; Wimmer et al., 1997, 2007).
The work presented here, is a part of pest management
program carried out in our laboratory. The main purpose
of present investigation is to explore the IGRs activites of
synthesized sulfonamides in comparison to in use IGRs
against G. mellonella (wax comb) under the laboratory
conditions. In this paper, we report designing, synthesis,
characterization, and in vivo analysis of JHAs having sul-
fonamide and amide feature along with hydrophobic
terminations at both the ends of the main chain
(Figure 1(c)). All analogs have been further screened for
their IGRs and pesticidal activity against G. mellonella.
been used for present study to predict the mode of bind-
ing of JH analogs to a receptor protein. Semiempirical
force field evaluates binding in two steps. In the first
step, the intramolecular energetics are estimated for the
transition from unbound states to the bound state
whereas second step evaluates the intermolecular ener-
getics of combining the ligand and protein in their bound
conformation. Analogs are screened based upon their
scoring function.
2.2. Preparation of files
The structures of JHAs have been built using pymol soft-
ware tool (www.pymol.com) and optimized using
AMBER force field. Gasteiger charge has been assigned
to ligands and then non-polar hydrogens are merged. The
rigid roots were defined automatically for each compound.
Further Auto Tor module has been applied in order to
define the flexibility of the bonds in the ligands. The PDB
file of the JHBP of G. mellonella (2RCK) has been down-
loaded from the protein data bank (www.rcsb.org). Polar
hydrogens were added to the macromolecule (2RCK)
using the ADDSOL utility of AutoDock 4.2. The Kollman
charges were added to each atoms of the chain A and
saved as protein.pdbqt format. Minimization was carried
out under vacuum medium. Electrostatic interactions were
calculated using the cut-off method. The solvation
parameters were added using the ADDSOL utility of Auto
Dock 4.2. Default values of atomic solvation parameters
were used throughout the calculations. Grid box was
generated around the binding cavity of the JHBP by
selecting key amino acid residues THR 22, TYR 128 and
130 (Kolodziejczyk et al., 2008) (Figure 2). Since the
structure of the protein kept rigid and known, interaction
energies between the probe and surrounding amino acids
2. Materials and methods
2.1. Principle of molecular docking using AutoDock 4.2
AutoDock 4.2 automated docking tool (The Scripps
Research Institute La Jolla, CA 92037-1000, USA) has
Figure 2. Complete structure of the binding protein (PDB 2RCK) (structures have been drawn using Autodock4.2 software).

4
P. Sharma et al.
has been calculated at each point in the grid and stored in
output file as pro.glg. The grids were chosen to be suffi-
ciently large so as to include not only the active site but
also significant portion of the surrounding surface at
receptor protein with grid points 69 × 67 × 69, along with
grid spacing of .531 Ǻ.
Lamarckian genetic algorithm (LGA) protocol has
been used for protein fixed: ligand flexible model
(Jenwitheesuk & Samudrala, 2003; Morris et al., 1998).
The scoring function is a sum of van der Waals,
hydrogen bonding, and distance-dependent dielectric
electrostatics as well as conformational torsional restric-
tion entropy and empirical solvation energetic in terms
of ligand–protein complex.
Docking calculations began with an initial population
of 150 randomly placed conformation of ligands. There-
fore, in totality hundred search attempts (ga_run parame-
ter) were performed for each ligand. The maximum
number of energy evaluations and generations before the
termination of LGA run were 2.5 × 106 and 2.7 × 104,
respectively. For the local search, the pseudo-solis and
wets algorithm were applied. Other docking parameters
were set to default. Final docking orientations lying
within 2 Ǻ of the root-mean square deviation tolerance
of each other were represented as most favorable
conformation with low free energy of binding (ΔGb). All
analogs were ranked according to their binding free
energy (ΔGb in kcal/mol) and inhibition constant (Ki in
μM) at 298.15 K. Overall, free energy of binding (ΔGb)
was composed of a sum of free energies measured for
each analog pose and given as:
400 MHz spectrophotometer operating at room tempera-
ture in DMSO d6 as solvent. The electron spray ioniza-
tion-mass spectroscopy analyses were carried out in
positive ion modes using a Water Q of Micromass.
2.3.1. General procedure for the synthesis of
benzenesulfonyl valine acid chlorde (e) and p
toluenesulfonyl valine chloride (f)
A mixture of valine (11.7 g, .1 mol) and benzenesulfonyl
chloride ((a); 17.6 g; .1 mol), and NaOH solution (1 N,
200 ml) was stirred together at 65–70°C for four hours
(John, 2000; Robert & Robert, 1992). A clear solution
was obtained. The reaction mixture was cooled to 5 °C
and treated with concentrated HCl to make it slightly
acidic (pH 6.5). Benzenesulfonyl valine separated out as
white crystals. It was recrystallized from hot water to
give pure benzenesulfonyl valine ((b); 16.02 g; 69.95%;
m.p. 118–120°). Similarly p-toluenesulfonyl valine was
prepared ((d); 17.8 g; 73.25%; m.p. 128–138°). Further
benzenesulfonyl valine ((b); 1 g; .004 mol) was dis-
solved in dry benzene and an excess of freshly distilled
thionyl chloride (5.0 g) was slowly added to it and
stirred at room temperature for 20 mins. The reaction
mixture was gently refluxed for 3 h. The solvent and
excess of thionyl chloride were then distilled off under
reduced pressure to give acid chloride of benzenesul-
fonyl valine (e). Similar procedure was followed for the
preparation of p-toluenesulfonyl valine chloride (f).
These (e–f) were used for next reaction without further
purification.
ΔGb
=
Intermolecular
Energy + Internal
Energy + Torsional energy − Unbound system’s energy
2.3.2. Synthesis of N-(1-isopropyl-2-oxo-3-aza-butanyl)
benzene sulfonamide and related compounds (T3–T8)
Ki ¼ exp ððDGb ꢀ 1000Þ=ðR ꢀ TÞÞ
(where ΔGb is the binding energy (BE), R = 1.98719 cal,
and T = 298.15 (temperature in Kelvin)
Combination of Intermolecular + internal energy
formed “dock energy”, while intermolecular + torsional
energy formed “BE”. Same protocol was applied for all
analogs.
All simulations have been performed on Linux
operating system with system Properties (Intel(R)
Pentium(R) D CPU 2.80 GHz, 4.0 GB of RAM).
The synthesis of N-(1-isopropyl-2-oxo-3-aza-butanyl)
benzene sulfonamide and related compounds (T3–T8)
was accomplished along the following lines (Scheme-1)
(Supplementary Figure I).
The acid chlorides of benzene and toluene sulfonyl
valine prepared above were treated with methyl amine at
room temperature for four hours to give desired products
N-(1-isopropyl-2-oxo-3-aza-butanyl) benzene sulfon-
amide (T3) and N-(1-isopropyl-2-oxo-3-aza-butanyl)–
p-toluene sulfonamide (T4), respectively.
Likewise acid chlorides of benzene and toluene sul-
fonyl valine were treated with N, N-dimethyl amine at
room temperature for four hours to give desired products
N-(1-isopropyl-2-oxo-3-aza-3-N-methyl-butanyl) benzene
sulfonamide (T5) and N-(1-isopropyl-2-oxo-3-aza-3-
N-methyl-butanyl)–p-toluene sulfonamide (T6), respec-
tively. Similar reaction of these acid chlorides (e–f)
with N, N-diethyl-amine gave the desired products
N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene
sulfonamide (T7) and N-(1-isopropyl-2-oxo-3-aza-3-N-
2.3. General methods and material for synthesis
Solvents used were purchased from Sigma Aldrich of
99% purity grade. The progress of the reaction was
monitored by thin layer chromatography. Melting points
were determined on a hot-stage apparatus and are uncor-
rected. FT-IR spectra were recorded on Perkin Elmer
1600 spectrophotometer with the samples as compressed
1
KBr pellets ranging from 4000 to 400 cm−1. H and 13C
NMR spectra were recorded using a Brucker Avance

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
5
ethyl-pentanyl)-p-toluene sulfonamide (T8), respectively,
in good yields. The structures of final products (T3–T8)
were established by spectroscopic studies (Supplemen-
tary Table I).
seen at 1000 ppm from 4 to 10 h exposure period. 2 mL
volume of each has been used for in vivo study.
Larvae of fourth instar of G. mellonella were col-
lected and maintained for the treatment. Acetone solution
(2 mL) containing the compound was poured on the filter
paper in each petri dish and allowed to evaporate. Later,
the counted number of larvae to be treated was trans-
ferred to the petri dishes and after applying the different
concentrations of the analogs; mortality rate was noted
for each of the replication set. Each treatment involved
three replicates with each replicate containing 10 insects.
Same procedure has been applied for all synthesized
analogs (T3–T8) and in use IGRs (T1 and T2).
2.4. Rearing of the insect model
An artificial diet has been developed during the year
2012–2013 for mass rearing of G. mellonella in our lab-
oratory (temperature 27 1°C, relative humidity 65
5%, and 16:8 h scoto-photo-phase regime) and pre-
pared by well-defined method with some modifications
(Ashfaq, Al-Tememi, & Ahmed, 2005).
2.4.3. Statistical analysis
2.4.1. Analogs (T1–T8) tested for IGR action
For IGR action, complete data were subjected to analysis
of variance, the average larval mortality data were sub-
jected to probit analysis for calculating LC50 and LC90
values (Finney, 1971).
For pesticidal action, data were grouped according to
number of analogs (T1–T8), concentration (in ppm) and
exposure time (in hours) and then subjected to analysis
of variance. Further, data were statistically analyzed to
calculate the critical difference at p ≤ .05.
A laboratory reared colony of G. mellonella larvae were
used for IGR activity. Ten larvae of fourth instars were
kept in a 500 ml glass beaker. Diverse concentration of
all analogs (T1–T8) ranging from 10 to 100 ppm, in ace-
tone on w/v basis, has been used for testing against lepi-
dopteran insect species in order to show IGR effects.
The control mortalities were corrected using Abbott’s
formula (Abbott, 1925).
observed mortality intreatment ꢁ observed mortality in control
Corrected mortality ¼
ꢀ 100
100 ꢁ control mortality
number of dead larvae
Percentage mortality ¼
ꢀ 100
number of larvae introduced
The LC50 and LC90 were calculated from toxicity
data using probit analysis (Finney, 1971).
3. Results and discussion
3.1. In-silico JHBP–JHAs interactions
JHAs are small organic molecules that interact with JHBP
of the receptor protein i.e. G. mellonella. The affinity and
specificity of the ligand binding to receptor is determined
by molecular surface patterns, their chemical similarities,
structural complementarity, and also governed by non-
covalent bonds that are electrostatic in nature. The bal-
ance between these forces during docking resulted in the
energetically favored binding. The electrostatic interaction
was due to the attraction between polar functional groups
at the molecule and amino acid residues (C=O and NH
backbone groups) inside the binding cavity. Docking
identified the correct position of the ligand inside the
binding pocket of the receptor and also predicted affinity
level between the ligands and the receptor (Awasthi &
Sharma, 2012a, 2012b, 2013; Bhattacharjee, Gupta, &
Karle, 2000; Harada et al., 2011).
2.4.2. Analogs (T1–T8) tested for anti-JH activity
(pesticidal action)
Different concentration of commercial IGRs, i.e.
Pyriproxyfen and Fenoxycarb ranging from 1000 to
10,0000 ppm, in acetone on w/v basis, has been used for
testing against lepidopteran insect species in order to show
pesticidal effects (Amiri et al., 2012; Fahmy, 2012;
Ghoneim et al., 2012; Mojaver & Bandani, 2010). We
have prepared the concentrations ranging from 250 to
2000 ppm to check the immediate action of all the synthe-
sized analogs with lesser exposure period (in hours).
Synthesized analogs (T3–T8) and in use IGRs (T1 and T2)
did not show any visible change in the insect behavior up
to 750 ppm for 10 h of exposure period but changes were

6
P. Sharma et al.

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
7
Figure 3. (a) BE profile of IGRs (T1–T8); (b) energy varia-
tions (intermolecular, internal, and torsional energy) of all the
analogs with JH binding protein of G. mellonella.
In the present study, AutoDock program was used to
investigate the BE profile (BE (ΔGb) kcal/mol), inhibi-
tion constant (Ki in μM) of sulfonamide series (T3–T8)
along with in use IGRs (T1–T2) with JHBP of G. mel-
lonella (2RCK) (Figure 2). The energy profile has been
tabulated according to the BE and inhibitory constant
(Ki) (Table 1). Smaller the value of BE smaller will be
the inhibitory constant (Ki), and better would be the bio-
logical activity. All analogs illustrated low BE profile
upon interaction with JHBP of G. mellonella than
Fenoxycarb but higher than pyriproxyfen (in use IGRs).
Among all analogs, T4, T3, and T8 gave the lowest BE
profile (Figure 3(a)). Total internal energy of all analogs
has been compared. There occurred a large deviation in
the value of total internal energy of synthesized sulfon-
amide analogs (T3–T8) over commercial in use IGRs
(Figure 3(b)). Analogs showed fluctuating energy profile
which could be attributed due to structural variations at
the main chain (Figure 1(c)).
Torsional energy is associated with the degree of
freedom of the molecule. More is the degree of

8
P. Sharma et al.
Figure 4. (a) H-bond interactions of in use IGRs Pyriproxyfen and Fenoxycarb (T1 and T2) with JH binding protein of G. mellonella.
(JH analogs are shown in blue. Hydrogen bond interactions between amino acid residue and ligands are shown in green-colored
arrow); (b) H-bond interactions between the amino acid residues at the binding pocket of JHBP of G. mellonella and synthesized best
screened analog (T3–T8) (JH analogs are shown in blue. Hydrogen bond interactions between amino acid residue and analogs are
shown in green-colored arrows).
freedom more is the possibility of change in the
conformation inside the pocket. An increase in torsional
energy is indicated by more interactions of analog with
amino acid residues inside the binding cavity. Though
torsional energy and internal energy balanced each
other but overall there is an increase in BE profile
(Figure 3(a) and (b)). Upon comparing the torsional
energy values of all analogs with in use IGRs;
Pyriproxyfen (T1) with minimum torsional energy i.e.
0.60 Kcal/mol contributing towards the lowering of
overall BE profile of the molecule. Inhibitory constant
(Ki) varied in a similar manner as BE profile for all
the synthetic analogs and in use IGRs (T1–T2). Synthe-
sized analogs T4, T3, and T8 found to posses lower
value for inhibitory constant (Ki) in comparison to rest
of the analogs in the series (Table 1). Values of inhibi-
tion constant of these analogs found to be comparable
to Pyriproxyfen (T1).

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
9
Figure 5. (a) Basic structure of all the analogs; (b) superimposed structure of the analogs (T3–T8) before docking; (c) superimposed
structure of the analogs (T3–T8) after docking.
All analogs have been compared and analyzed. A uni-
form trend has been observed in BE sketch for the methy-
lated and non-methylated analogs (T3–T8). Analogs T3,
T5, and T7 being non-methylated and analogs T4, T6, and
T8 being methylated at ring A (Figure 1(c)). All methy-
lated analogs exhibited lower BE profile in comparison to
non-methylated analogs (Table 1). Analog T4 showed
lower BE followed by T3, T8, T7, T6, and T5 analogs,
respectively. Lower BE profile was due to the presence of
two different functional groups at the terminal ends of the
main chain which ultimately stretches the analog and pre-
vents the folding of the molecule inside the binding
pocket. Functional groups present at the main chain in
analog T4 was free for H-bonding interactions with amino
acid residues (Table 1) and exhibited strong H-bond
interactions with pocket residue THR 22 along with LYS
218. T3 analog also followed the same lines. In case of
T5 and T6, dimethyl group at the terminal end of the
chain induced steric hindrances; hence, none of the func-
tionality present at the main chain remained free for
forming H – bond with amino acid linings of binding
cavity. Only sulfonamide groups present at 2nd position
at main chain involved for H-bonding with LYS 218
(Table 1). In T7 and T8 analogs (diethyl amino group),
the length of the parent chain has been elongated and
molecules adopted the open terpenoid structure, which
could be the reason for their low BE profile.
On comparing all the analogs (T3–T8), it was con-
cluded that functionalities present at position 2nd, 5th,
and 6th involved effectively through H-bonding with
amino acid residues of the binding pocket. However,
functionality present at 3rd position exhibited the inert

10
P. Sharma et al.
Table 2. Additional interactions (bold – common, normal – additional) of the IGRs (in use T1–T2 and synthesized T3–T8) with
amino acid residues at the binding pocket of JHBP of G. mellonella.
S. No. IDs Additional interactions of the ligands with amino acid residues inside the binding pocket
1.
2.
3.
4.
5.
6.
7.
8.
T1 THR 22, LYS 85,218, PRO 146**, THR 148
T2 THR 22, TYR 130, LYS 218
T3 THR22, LYS 218, SER* 129, PRO146**, TYR 130, THR 148, ALA 21,220, GLU 147, HIS* 207, AGR** 210,214
T4 LYS 39, 218, THR 22, TYR 130, ALA 21,220, SER* 129, PRO** 146, HIS* 207, GLU 143,147, ARG** 210
T5 LYS 39, 218, SER* 129, HIS* 207, ARG** 210,214
T6 LYS 39, 218, THR 22, SER* 129, ARG** 210, 214, HIS* 207, GLU 147
T7 LYS 39,218, SER* 129, HIS* 207, ARG** 210,214,GLU 147
T8 LYS 39, 218, SER* 129, HIS* 207, ARG** 210,214, GLU 147
*Mixed codon.
**Strong codon.
T1: Pyriproxyfen (standard).
T2: Fenoxycarb (standard).
T3: N-(1-isopropyl-2-oxo-3-aza-butanyl) benzene sulfonamides.
T4: N-(1-isopropyl-2-oxo-3-aza-butanyl)-p-toluene sulfonamides.
T5: N-(1-isopropyl-2-oxo-3-aza-3-N-methyl-butanyl) benzene sulfonamides.
T6: N-(1-isopropyl-2-oxo-3-aza-3-N-methyl-butanyl)-p-toluene sulfonamides.
T7: N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene sulfonamides.
T8: N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl)-p-toluene sulfonamides.
Table 3. Probit analysis for IGR activity of all the analogs (T1–T8) against G. mellonella.
95%
% Larval mortality
Conc (ppm)
Confidence
95% Confidence
Limits
Limits
S. No. Analogs 10 25 35 50 100 LC50 (ppm)
LCL
UCL
LC90 (ppm)
LCL
UCL
Regression
1.
2.
3.
4.
5.
6.
7.
8.
T1
T2
T3
T4
T5
T6
T7
T8
44 59 67 78
46 61 68 79
25 32 50 64
26 37 52 67
29 36 50 57
21 32 46 68
25 32 50 64
32 50 64 71
89
89
78
82
71
82
79
82
9.99
10.12
24.76
30.73
38.45
34.15
34.14
19.48
2.44 17.53
3.69 16.54
16.50 33.01
17.96 43.51
9.96 66.94
21.40 46.90
18.51 49.77
9.48 29.48
153.27
131.69
112.15
191.46
427.02
167.13
217.10
172.00
75.00 231.54 3.53 + 1.25X
73.22 190.17 3.52 + 1.30X
81.46 142.84 2.16 + 2.00X
113.46 269.45 2.56 + 1.62X
106.10 747.94 3.09 + 1.21X
108.68 225.58 2.10 + 1.87X
118.72 315.48 2.52 + 1.60X
96.06 247.95 3.09 + 1.42X
T1: Pyriproxyfen (standard).
T2: Fenoxycarb (standard).
T3: N-(1-isopropyl-2-oxo-3-aza-butanyl) benzene sulfonamides.
T4: N-(1-isopropyl-2-oxo-3-aza-butanyl)-p-toluene sulfonamides.
T5: N-(1-isopropyl-2-oxo-3-aza-3-N-methyl-butanyl) benzene sulfonamides.
T6: N-(1-isopropyl-2-oxo-3-aza-3-N-methyl-butanyl)-p-toluene sulfonamides.
T7: N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene sulfonamides.
T8: N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl)-p-toluene sulfonamides.
behavior i.e. did not take part in H-bonding with pocket
residues. More is the formation of H-bonding of the
ligand with in pocket residues, stronger will be the pro-
tein–ligand complex (Figure 4(a) and (b)). Therefore,
analogs adopted different conformation inside the pocket
in order to have maximum number of H-bond with
amino acid residues of the binding pocket in docked
state (Figure 5(a)–(c)). The synthesized analogs (T3–T8)
have shown major interactions with amino acid residues
LYS 218, THR 22, and TYR 130 of the receptor protein
(Table 1). In T1 (Pyriproxyfen) functional group present
at position 2nd and 5th at main chain show H-bonding
with THR 22. Pyridine ring present in T1 showed addi-
tional H-bond interaction with PRO 146 amino acid resi-
due of the pocket. In T2 (Fenoxycarb), functional group
present at 6th position show H-bonding with LYS 218. It
also showed additional interactions with THR 22, TYR
130 which could be the reason for their good BE behav-
ior over synthesized analogs with JHBP of G. mellonella
(Figure 4(a)). Likewise analog T3 and T4 showed major
interactions with THR 22, TYR 130 key pocket residues
(Figure 4(b)). Synthesized analogs (T5–T8) showed H-
bonding interactions with amino acid residues – PRO
146, SER 129, HIS 207, ARG 210, 214 at binding
pocket which confirms concluded the hydrophobic,
acidic, and basic nature of the binding pocket (Table 2).
The residues which involved for additional interac-
tions have been referred as strong codon for effective

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
11
Figure 6. Concentration–mortality response of different analogs to fourth instar larvae of G. mellonella by topical application method.
binding. They all had the strong tendency to form hydro-
gen bonds with the ligand molecules (Wilhelm &
Nikolajewa, 2004). These results are supported by
spectroscopic studies of binding of JH-II with JHBP of
G. mellonella (Krzyżanowska, Lisowski, & Kochman,
1998). The idea of combination of multi-functionality
like sulfonamide functionality as well as amino acid
groups at main chain with aromatic closing at one termi-
nal along with substituted amine groups have been done
in order to have enhanced intermolecular interactions at
binding site (Figure 5(a)). Interaction energy (Hydrogen
bond, hydrophobic interactions, and van der Waals) has
been computed in terms of groups present on the surface
of the receptor and the ligand. The type and magnitude
of these interactions influenced the complex (JHAs –
JHBPs) stability. Variations in these properties near the
active site of the proteins were the main factor toward
selectivity and specificity. Elongated hydrocarbon chain
lengths reported to have improved JH activities (Mori
et al., 1975; Wheeler & Nijhout, 2003). Moreover, the
incorporation of free amine groups as well as sulfon-
amide group at the main chain also increased the
insecticidal activity (Ali, Nassar, Badawi, & Afify, 2010;
Tsikolia et al., 2013). Addition of the aromatic ring at
one terminal increased the hydrophobic character of the
analog, therefore enhanced π–cation interactions stabilize
the analogs inside the pocket (Figure 4(b)). Closer the
packing of the analog inside the pocket, stronger be the
interaction of JHAs with JHBP, which ultimately lowered
BE profile. Combinations of different functionalities led
to active change in conformation of JHAs inside the
binding cavity in comparison to JHAs outside the

12
P. Sharma et al.

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
13
Figure 7a. Physiological changes on G. mellonella larvae after applying 2000 ppm concentrations of N-(1-isopropyl-2-oxo-3-aza-3-
N-ethyl-pentanyl)-p-toluene sulfonamide (T8) at exposure period of 2 h in comparison to Pyriproxyfen (T1) and Fenoxycarb (T2).
binding pocket and responsible for the variation in BE
3.3. Bio assay of synthesized analogs on G.
profile (Figure 5(a)–(c)) (Goodman, Schooleyt,
&
mellonella
Gilbert, 1978; Koeppe & Kovalick, 1986).
All analogs have been screened for their IGRs and
pesticidal action against fourth instar larvae of G.
mellonella (Honey comb pest). Population of 10 larvae
each in three replication sets have been studied for larval
mortality at different concentrations (in ppm) having
variable exposure periods (in hours).
Table 3 provides the results of IGR action of all
the synthesized analogs (T3–T8) along with in use
IGRs (T1 and T2) during the treatment of fourth instar
of G. mellonella at different concentrations (10–
100 ppm). Commercial IGRs (T1–T2) showed the
highest mortality at concentrations ranging from 10 to
100 ppm after 72 h of exposure period of time. LC50
value for T1 and T2 was 9.99 and 10.12 ppm,
3.2. Synthesis of N-(1-isopropyl-2-oxo-3-aza-butanyl)
benzene sulfonamide and related compounds (T3–T8)
Synthesis of N-(1-isopropyl-2-oxo-3-aza-butanyl) ben-
zene sulfonamide and related compounds (T3–T8) has
been achieved by the action of substituted amines with
acid chlorides in dry benzene. N-(1-isopropyl-2-oxo-3-
aza-butanyl) benzene sulfonamide and related com-
pounds (T3–T8) have been found to be new in literature
and fully identified on the basis of their spectral data
(IR, 1H NMR, 13C NMR, and ESI-MS analysis).
All compounds have been obtained as pure solids
(Supplementary Table I).

14
P. Sharma et al.
Figure 7b. Physiological changes on G. mellonella larvae after applying 1500 ppm concentrations of N-(1-isopropyl-2-oxo-3-aza-3-
N-ethyl-pentanyl)-p-toluene sulfonamide (T8) at exposure period of 4 h in comparison to Pyriproxyfen (T1) and Fenoxycarb (T2).
respectively. Among the synthesized analogs (T3–T8),
analog T8 exhibited the highest mortality rate having
LC50 19.48 ppm at these concentrations ranging from
10 to 100 ppm after 72 h of exposure period of time
(Table 3 and Figure 6).
None of the treatments was significantly effective at
1000 ppm concentration after exposure of 2 h. Although
there was a progressive increase in mortality with
increase in exposure period, but overall rate of mortality
remained low at this concentration. T1 showed maximum
efficacy of 100.00 kills at 8 h exposure period followed
by mortality of 100.00% by T2 at same concentration
and exposure time. T4, T6, and T8 with same concentra-
tion exhibited 93.33, 76.67, and 73.33% mortality,
respectively, at the exposure period of 8 h. Mortality did
not exceed beyond 65.00 % in rest of the treatments for
8 h exposure period. Increasing the exposure periods up
to 10 h, T4, T6, and T8 attained the 100.00% larval mor-
tality followed by T5 and T7 at the same concentration.
Mortality did not exceed beyond 70.00% in T3 formula-
tions at the same exposure period.
Trends were similar at 1500 ppm concentration. T7
formulation was found to be least effective with 10.00%
kill at 2 h exposure time while T6 formulation at the
same exposure period exhibited the 30.00% mortality.
While percent mortality in T6 enhanced from 30.00% at
2 h exposure to 76.67% at 6 h, T2 showed the enhance-
ment from 53.33 to 100.00% at 6 h followed by T1 from
46.67 to 100.00% mortality at similar exposure periods.
Further, pesticidal action of all the analogs (T1–T8)
has been studied at higher concentrations ranging from
750 to 2000 ppm at different exposure period of time
from 2 to 10 h. No mortality rates have been seen at low
concentrations (750, 500, and 250 ppm) with respect to
exposure period (2–10 h). The larval mortality of G.
mellonella increased with increase in concentration of
the different analogs based formulations. A significantly
progressive increase in mortality was recorded with the
increase in exposure period as well as concentration
(Supplementary Figure II). All treatments showed maxi-
mum efficacy against G. mellonella at 2000 ppm after
exposure of 10 h. All the synthetic analogs (T3–T8)
showed significant effects on larval mortality at 1000
and 1500 ppm after 10 h exposure. The observations
regarding effect of different concentrations of sulfon-
amide IGR’s at different exposure periods on percent lar-
val mortality of G. mellonella have been tabulated in
Table 4.

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
15
Figure 7c. Physiological changes on G. mellonella larvae after applying 1000 ppm concentrations of N-(1-isopropyl-2-oxo-3-aza-3-
N-ethyl-pentanyl)-p-toluene sulfonamide (T8) at exposure period of 6 h in comparison to Pyriproxyfen (T1) and Fenoxycarb (T2).
T8 also showed the increase from 16.67 to 70.00% kill.
Likewise, T4 exhibited the enhancement from 16.67 to
80.00% kill at similar exposure period (6 h) at same con-
centration. In case of T7 formulation 86.67 % larvae
were killed when exposed to 1500 ppm for 6 h. T3 and
T5 were least effective registering 53.33 and 60.00%
mortality and these values were insignificant to each
other (Table 4).
Highest percent of larval mortality at 2000 ppm con-
centration up to 4 h exposure was shown by T1
(100.00%), T2 (100.00%) followed by T8 and T7. Like-
wise T6, T4, and T3 also showed higher mortality of
96.67, 93.33, and 93.33%, respectively, at the same
exposure period of 4 h at same concentrations. Formula-
tion T5 showed the least 90.00% mortality which is
statistically insignificant to others. However, at 6 h expo-
sure, rest of the formulations showed the 100.00% larval
mortality. Overall there occurred the increase in mortality
with increase in concentration and increase in exposure
period. Clear morphological changes have been observed
at 4th larval stage of G. mellonella (Figure 7a–c).
Results showed that formulation T8 to be the most
effective sulfonamide formulation at all concentrations
closely followed by T7 which proved to be more effec-
tive at the highest concentration of 2000 ppm at 4 h
exposure period. T6 though less effective than T8 and T7
showed significantly improved efficiency against
G. mellonella larvae as compared to T4, T3, and T5. The

16
P. Sharma et al.
Figure 8. Comparison of in silico study and in vivo screening (at 1500 ppm for 6 h and at 2000 ppm for 4 h).
mortality rate achieved by T4, T5, T6 at 2000 ppm con-
centration after 4 h exposure (93.33%) was significantly
similar to that achieved by T8 at similar exposure period
but at low concentration of 1500 ppm (Figure 7(a)–(c)).
Pyriproxyfen (IGRs). Among synthesized series (T3–T8);
analog T4 found to have the lowest energy profile fol-
lowed by T3 and T8 analogs. Methylated analog (T4)
having free amine group at terminal R2 position exhib-
ited lowest BE in comparison of the non-methylated ana-
logs (T3). Overall, all synthesized analogs (T3–T8)
showed BE in the range of ΔGº ≥ 2 kcal/mol; suggested
to be effective IGR’s in comparison to in use (T1 and
T2) IGRs. Further, bioassay of T3–T8 along with com-
mercial in use T1 and T2, on G. mellonella has been car-
ried out with increase in concentration as well as
increase in exposure period. Sulfonamide formulations
used in our experiment are rated as T8 > T4 > T7
> T6 > T5 ~ T4 > T3. Interestingly, bio assay results are
being supported by theoretical outcome (Figure 8).
As per literature review and work done by other
groups regarding nature of the inner lining of binding
cavity of JHBP and structure of naturally occurring JH’s
(J-I, JH-II, JH-III); it is the hydrophobic nature of the
cavity and dominating hydrophobic structure of the JH’s
played vital role in binding process and further signaling
for the physiological action of complex. But due to the
unsaturation at the main chain of JH along with hetero
atoms dictates the specificity of binding of JH to receptor
protein. Number of analogs of JH has been synthesized
and evaluated based on these lines. We worked on
incorporating the design features which are very com-
mon in other bio-applications like – sulfonamide and
amides in the main chain and incorporated the hydropho-
bic groups at three corners of the molecule (Figure 5(a)).
Aromatic substitution at A ring, and sulfonamide func-
tionality initiates/stabilizes the binding process but finally
it is amide along with hydrophobic termination responsi-
ble for effective binding (Unpublished results Awasthi
et al.). Present study concludes the sulfonamide’s series
to be effective IGR’s at par to commercially in use –
Pyriproxyfen and Fenoxycarb. Detail study on stability/
degradability is to be carried for field application of the
analogs and its environmental effect in comparison to
A
comparison between bio assay and virtual
screening has been shown in Figure 8. The theoretical
trend for BE followed the pattern: T2 > T5 >
T6 > T7 > T8 > T3 > T4 > T1. The BE values for all the
analogs occurred within the range of 2 kcal/mol. These
computational finding clearly showed the similar behav-
ior of synthesized analogs in comparison to in use IGRs
(T1 and T2) against JHBP of G. mellonalla. These results
were further supported by in vivo studies. Analogs T6–
T8 at 2000 ppm at 4 h of exposure period showed a
similar activity that of T1 and T2 by yielding 100% mor-
tality. Further decreasing concentration to 1500 ppm at
8 h exposure period 100% mortality has been achieved
by T4, T6–T8. Thus, analog T8 having leading behavior
over T1 and T2 as per in vivo study. Overall study
confirms that all synthesized analogs (T3–T8) qualified to
be potential IGRs as supported by computational and
biological results.
4. Conclusions
The proposed work is a part of designing and synthesis
of effective and environmentally safe JH analogs to be
IGR. We have computationally designed the analogs
based upon their interaction with JHBP of G. mellonella
and synthesized the same series. Molecular docking sug-
gested that JH analogs (T3–T8) with minimum BE fit
better inside the binding pocket of the receptor protein.
The crystal structure of JHBP of G. mellonella has been
taken from protein data bank (Kolodziejczyk et al.,
2008). Six synthetic analogs reported in this paper differ
from one another based on substituent groups attached at
aromatic ring A and aliphatic termination at R2. Synthe-
sized JHAs (T3–T8) exhibited lower BE profile as com-
pared to Fenoxycarb (IGRs), but higher than

In silico and bio assay of juvenile hormone analogs as an insect growth regulator
17
Awasthi, P., & Sharma, P. (2013). Designing and binding
mode prediction of juvenile hormone analogs as potential
inhibitor for Galleria mellonella. Journal of Computer
Science and Systems Biology, 6, 106–111.
Bhattacharjee, A. K., Gupta, R. K., & Karle, J. M. (2000).
Molecular similarity analysis between insect juvenile hor-
mone and N, N-diethyl-m-toluamide (DEET) analogs may
aid design of novel insect repellents. Journal of Molecular
Recognition, 13, 213–220.
presently in use IGR’s and work will be published
subsequently. Present work is a new and fast approach to
design and develop new class of IGR’s and its applica-
bility to other lepidopteron insect’s species. Besides,
further investigation regarding the effect on non-target
organism is extremely important and imperative in the
near future.
Bortolotti, L., Porrini, C., Sbrenna, A. M., & Sbrenna, G.
(2000). Ovicidal action of fenoxycarb on a predator,
Chrysoperla carnea (Neuroptera: Chrysopidae). Applied
Entomology and Zoology, 35, 265–270.
Supplementary material
The supplementary material for this paper is available
Devillers, J. (Ed.). (2013). Juvenile hormone and juvenoids:
Modeling biological effects and environmental fate. France:
CRC Press.
online
1056549.
at
http://dx.doi.org/10.1080/07391102.2015.
Fahmy, N. M. (2012). Impact of two insect growth regulators
on the enhancement of oxidative stress and antioxidant effi-
ciency of the cotton leaf worm. Egyptian Academic Journal
of Biological Sciences, 5, 137–149.
Finney, D. J. (1971). Probit analysis. London: Cambridge
University Press.
Frings, M., Thomé, I., & Bolm, C. (2012). Synthesis of chiral
sulfoximine-based thioureas and their application in
asymmetric organocatalysis. Beilstein Journal of Organic
Chemistry, 8, 1443–1451.
Fujimoto, Z., Suzuki, R., Shiotsuki, T., Tsuchiya, W., Tase, A.,
Momma, M., & Yamazaki, T. (2013). Crystal structure of
silkworm Bombyx mori JHBP in complex with 2-Methyl-2,4-
Pentanediol: Plasticity of JH-binding pocket and ligand –
Induced conformational change of the second cavity in JHBP.
PLoS ONE, 8, e56261. doi:10.1371/journal.pone.0056261
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tance on the JH action, introductory paper No. 168.
Sweden: Lund University.
Acknowledgment
Authors thank Sophisticated Analytical Instrument Facility,
Punjab University, Chandigarh, for recording FT-IR, NMR (1H
and 13C) and mass spectra. We are also thankful to Director,
Institute of Biotechnology and Environmental Science – Neri
(Hamirpur) HP for carrying out biological evaluation of the
analogs. One of the authors Priyanka Sharma acknowledges
Ministry of Human Resources Development, Government of
India for PhD fellowship.
Disclosure statement
No potential conflict of interest was reported by the authors.
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