Anthelmintic activity of a nanoformulation based on thiophenes identified in Tagetes patula L. (Asteraceae) against the small ruminant nematode Haemonchus contortus

Article abstract of DOI:10.1016/j.actatropica.2021.105920

The synthesis of thiophenic compounds, previously identified in Tagetes patula, revealed that 4-(5′-(hydroxymethyl)-[2,2′-bithiophene]-5-yl)but-3-yn-1-ol), or simply Thio1, has a pronounced in vitro anthelmintic effect against Haemonchus contortus, showing 100% efficacy in the egg hatch and larval development tests presenting EC₅₀ = 0.1731 mg.mL^?1 and EC₅₀ = 0.3243 mg.mL^?1, respectively. So, this compound was selected to preparation of a nanostructured formulation to be orally administered to Santa Inês sheep. In general, from the fecal egg count reduction test (FECRT), it was observed that the product kept the parasitic load in the digestive tract of the hosts stable, with eggs per gram of faeces (EPG) counts having a mean value < 3,000 (EPG_mean = 2167.1, efficacy = 36,45%), thus protecting the animals from health risks caused by a massive nematode infestation. To better understand the mode of action of this thiophene derivative, in silico molecular modeling studies were carried out with the glutamate-activated chloride channel (GluCl), a well-known molecular target of anthelmintic compounds. Based on the affinity score (GlideScore = -5.7 kcal.mol^?1) and the proposed binding mode, Thio1 could be classified as a potential GluCl ligand, justifying the promising results observed in the anthelmintic assays.

Full text of DOI:10.1016/j.actatropica.2021.105920

Acta Tropica 219 (2021) 105920
Contents lists available at ScienceDirect
Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
Anthelmintic activity of a nanoformulation based on thiophenes identified
in Tagetes patula L. (Asteraceae) against the small ruminant nematode
Haemonchus contortus
Fl ´a vio Augusto Sanches Politia,*, Renata Vieira Bueno , Lucas Andr ´e Zeoly ,
b
c
a
d
a
c
Rafaela Regina Fantatto , Josimar de Oliveira Eloy , Marlus Chorilli , Fernando Coelho ,
Rafael Vict o´ rio Carvalho Guido , Ana Carolina de Souza Chagas , Maysa Furlan
b
e
f
a
School of Pharmaceutical Sciences, S a˜ o Paulo State University (UNESP), Araraquara (SP), Brazil
University of S a˜ o Paulo (USP), Physics Institute of S a˜ o Carlos (IFSC), S a˜ o Carlos (SP), Brazil
b
c
University of Campinas (UNICAMP), Chemistry Institute, Campinas (SP), Brazil
College of Pharmacy, Dentistry and Nursing, Federal University of Cear a´ (UFC), Fortaleza (CE), Brazil
Brazilian Agricultural Research Corporation (EMBRAPA), Southeast Livestock, S a˜ o Carlos (SP), Brazil
Chemistry Institute, S a˜ o Paulo State University (UNESP), Araraquara (SP), Brazil
d
e
f
A R T I C L E I N F O
A B S T R A C T
′
Keywords:
The synthesis of thiophenic compounds, previously identified in Tagetes patula, revealed that 4-(5 -(hydrox-
Docking
′
ymethyl)-[2,2 -bithiophene]-5-yl)but-3-yn-1-ol), or simply Thio1, has a pronounced in vitro anthelmintic effect
French marigold
Gastrointestinal nematode
in vitro
against Haemonchus contortus, showing 100% efficacy in the egg hatch and larval development tests presenting
ꢀ 1
ꢀ 1
EC50 = 0.1731 mg.mL and EC50 = 0.3243 mg.mL , respectively. So, this compound was selected to prepa-
ration of a nanostructured formulation to be orally administered to Santa In ˆe s sheep. In general, from the fecal
egg count reduction test (FECRT), it was observed that the product kept the parasitic load in the digestive tract of
the hosts stable, with eggs per gram of faeces (EPG) counts having a mean value < 3,000 (EPGmean = 2167.1,
efficacy = 36,45%), thus protecting the animals from health risks caused by a massive nematode infestation. To
better understand the mode of action of this thiophene derivative, in silico molecular modeling studies were
carried out with the glutamate-activated chloride channel (GluCl), a well-known molecular target of anthel-
mintic compounds. Based on the affinity score (GlideScore = -5.7 kcal.molꢀ ) and the proposed binding mode,
Thio1 could be classified as a potential GluCl ligand, justifying the promising results observed in the anthelmintic
assays.
in vivo
UNC-49 receptor
1
1
. Introduction
Haemonchus spp. nematodes (phylum Nemathelminthes, class Nem-
not show clinical signs, subclinical infections are also a major concern
for producers owing to weakness and significant decreases in weight,
fertility and milk production (Mir et al. 2007). The main control strategy
relies on synthetic anthelmintics, whose targets are diverse proteins,
including β-tubulin, acetylcholine receptors, estrogenic receptors, and
receptors involved in synaptic neurotransmission, called Cys-loop
ligand-gated, which block chloride channels [LGCCs] (Kohler 2001).
However, their indiscriminate use has resulted in the selection of par-
asites insensitive to available products.
atoda, order Strongylida, family Trichostronglylidae) were first
described in 1803 by Karl Rodolphi (Soulsby 1982). Twelve species of
this helminth were characterized, of which three, H. contortus, H. placei
and H. similis, are globally found parasitizing sheep, goats and cattle
(
Hoberg et al. 2004). The larval and adult stages of H. contortus, due to
its hematophagy, generate huge economic losses in sheep farming,
causing from severe anemia to the death of heavily infected animals,
characterizing a disease known as hemonchosis (Bethony et al. 2006).
Although animals with low levels of infection within a flock generally do
This current prospect of parasites highly resistant to available
chemicals – including benzimidazoles (BZs), imidazothiazoles, macro-
cyclic lactones and the recently introduced amino-acetonitrile
*
Corresponding author. Tel.: (+55) 16-3301-6961. ORCID number: 0000-0001-6321-5509.
E-mail address: flaviopoliti@hotmail.com (F.A.S. Politi).
https://doi.org/10.1016/j.actatropica.2021.105920
Received 22 September 2020; Received in revised form 2 February 2021; Accepted 7 April 2021
Available online 20 April 2021
0
001-706X/© 2021 Elsevier B.V. All rights reserved.

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
derivative class – in addition to the toxicity of these products to humans
and treated animal, as well as its cumulative effect on the environment,
has emphasized the research and discovery of new chemical and bio-
logical alternatives to control the disease (Kotze and Prichard 2016). In
this sense, recent works attest to the parasiticide potential of plant
derived from Tagetes patula L. (Asteraceae), popularly known as French
2. Material and methods
′
2.1. Experimental procedure for the synthesis of 5-bromo-2,2 -
bithiophene
′
marigold, an annual plant widely distributed in both hemispheres.
Phytochemical analyses of this plant species point to the presence of
important classes of secondary metabolites, such as flavonoids (Vasu-
devan et al. 1997, Politi et al. 2012, Al-Musayeib et al. 2014), alkaloids
A stirred solution of 2,2 -bithiophene (1.66 g, 9.98 mmol, 99% pu-
rity, Sigma-Aldrich, USA) in chloroform/acetic acid (reagent grade, 1:1,
◦
60 mL, 0.17 M, 1.0 equivalent, Sigma-Aldrich, USA) was cooled to 0 C,
under a nitrogen atmosphere. N-Bromo-succinimide (1.78 g, 9.98 mmol,
99% purity, 1.0 equivalent, Sigma-Aldrich, USA) was added in small
portions to the reaction, and after the end of the addition, the mixture
(
Faizi and Naz 2002), benzofurans (Sutfeld et al. 1985, Tang et al. 1987,
Parodi et al. 1988, Menelaou et al., 1991; Politi et al., 2018) and thio-
phenes (Vasudevan et al., 1997; Bano et al., 2002; Marotti et al., 2010).
These latter are unsaturated heterocyclic compounds with
five-membered rings containing sulfur as a heteroatom. The genus
Tagetes is a natural source for its obtention, having already described
◦
was allowed to reach room temperature (25 C) and then stirred for 12
hours. The reaction mixture was poured into cooled water (100 mL) and
extracted with chloroform (reagent grade, 75 mL, Sigma-Aldrich, USA).
The organic layer was washed with saturated bicarbonate solution (2 ×
100 mL) and brine (1 × 100 mL), dried over anhydrous sodium sulfate,
and finally evaporated under reduced pressure. The crude product was
′
several of these compounds: 5-(3,4-diacetoxy-l-butinyl)-2,2 -bithio-
′
phene (Menelaou et al. 1991); 5-(3-buten-1-ynyl)2,2 -bithiophene
(
Margl et al. 2002); methyl-5-[4-(3-methyl-1-oxobutoxy)-1-butynyl]-2,
purified using flash column chromatography (SiO
2
[100:1, silica:crude
′
2
2
bithiophene (Bano et al. 2002); 5-methyl-2,2’,5’,2’”,5’’,2’’’,5’’’,
mass], hexane) to provide product 1a as a light green viscous oil (1.45 g,
1
’’’-quinquethiophene (Al-Musayeib et al. 2014); thiotagetin (Ibrahim
59% yield). H NMR (250 MHz, CDCl
3
) δ 7.25 (dd, J = 5.1 Hz, 1.2 Hz,
′
and Mohamed 2016) and 5-(4-hydroxybut-1-ynyl)2,2 -bithiophene
1H), 7.15 (dd, J = 3.6 Hz, 1.2 Hz, 1H), 7.04 (dd, J = 5.1 Hz, 3.6 Hz, 1H),
7.00 (d, J = 3.8 Hz, 1H), 6.95 (d, J = 3.8 Hz, 1H).
(
Politi et al. 2018).
Thus, the aim of this work was to evaluate the anthelmintic efficacy
of synthetic thiophene derivatives inspired by natural products extrac-
2
.2. Experimental procedure for the synthesis of 4-([2-2’-bithiophen]-5-
ted from T. patula in the in vitro assay against the multiresistant isolate of
yl)-but-3-in-1-ol
H. contortus Embrapa 2010, and also, evaluate the in vivo anthelmintic
activity of a nanoformulation prepared with the most active thiophene
selected from the results obtained in the in vitro assays in sheep artifi-
cially infected by the nematode. Many promising compounds researched
lately are unstable and may suffer reactions that lead to decreased or lost
efficiency and even product degradation. An alternative to increase
stability and allow controlled release is the encapsulation of active
substances through techniques involving nanotechnology. Nano-
encapsulation consists of compartmentalization of substances in poly-
meric carriers, with size ranging between 50 and 300 nm. Finally, an in-
silico study was performed in order to understand a possible mechanism
of action of the selected compound into the ivermectin binding site of
the H. contortus glutamate-gated chloride (GluCl) homology model. The
GluCl channels are pentameric receptors for the inhibitory neurotrans-
mitter glutamate in invertebrates and are a major target for macrocyclic
lactones anthelmintics.
Compound 1a (245.2 mg, 1.00 mmol, 1.0 equivalent), copper iodide
(19.0 mg, 0.10 mmol, 98% purity, 0.1 equivalent, Sigma-Aldrich, USA),
palladium acetate [Pd(OAc)
2
] (22.4 mg, 0.10 mmol, 98% purity, 0.1
(CO )] (976.8
equivalent, Sigma-Aldrich, USA), cesium carbonate [Cs
2
3
mg, 3.00 mmol, 99% purity, 3.0 equivalents, Sigma-Aldrich, USA) and
Xantphos (115.6 mg, 0.20 mmol, 97% purity, 0.2 equivalent, Sigma-
Aldrich, USA) were added to a two-necked round bottom flask. A
reflux condenser was connected to the flask, and the system was purged
with nitrogen for 15 minutes. Degassed dimethylformamide (DMF, 4.0
mL 0.25 M, Supelco®) was added, and to the obtained suspension, 3-
butin-1-ol (98.3
μ
L, 1.30 mmol, 1.3 equivalents) was slowly added.
◦
The reaction mixture was heated to 80 C and stirred for 3 hours. After
that time, it was diluted with ethyl acetate (30 mL) and washed with
hydrochloric acid (HCl 1 M, 20 mL), distilled water (20 mL), and brine
(20 mL), dried over anhydrous sodium sulfate, evaporated under
reduced pressure and purified by flash column chromatography [SiO
2
,
hexane:ethyl acetate (70:30 v/v)] to provide 1b as a yellow solid (214.2
1
mg, 91% yield). H NMR (250 MHz, CDCl
3
) δ 7.22 (dd, J = 5.1, 1.1 Hz,
2

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
1
4
H), 7.16 (dd, J = 3.6, 1.1 Hz, 1H), 7.05 (d, J = 3.8 Hz, 1H), 7.00 (t, J =
.1 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H), 2.72 (t, J = 6.3 Hz, 2H), 1.98 (s, 1H)
under a nitrogen atmosphere. Then, N-iodosuccinimide (NIS, 752.75
mg, 3.35 mmol, 95% purity, 1.3 equivalents, Sigma-Aldrich, USA) was
added in small portions, and the resulting mixture was allowed to reach
1
3
C NMR (63 MHz, CDCl
3
) δ 138.03, 136.74, 132.39, 127.87, 124.83,
◦
1
24.09, 123.27, 122.09, 91.63, 75.50, 60.96, 24.16.
room temperature (25 C) and stirred in the dark for 12 hours. The sol-
vent was evaporated under reduced pressure, and the crude product was
recrystallized from hot ethanol and then filtered and dried under vac-
uum to provide product 2b as a yellow amorphous solid (424.0 mg, 51%
1
yield). H NMR (250 MHz, DMSO-d6) δ 9.87 (s, 1H), 7.95 (d, J = 4.0 Hz,
1
H), 7.47 (d, J = 4.0 Hz, 1H), 7.38 (d, J = 3.8 Hz, 1H), 7.28 (d, J = 3.8
′
2
.3. Experimental procedure for the synthesis of [2,2 -bithiophene]-5-
Hz, 1H).
.5. Experimental procedure for the synthesis of 5 -(4-hydroxybut-1-in-1-
carbaldehyde
′
2
′
yl) [2,2 bithiophene]-5-carbaldehyde
To a two-necked round-bottom flask, compound 2b (132.2 mg, 0.41
mmol, 1.0 equivalent), copper iodide (7.9 mg, 0.041 mmol, 0.1 equiv-
2
alents), Pd(OAc) (4.63 mg, 0.021 mmol, 0.05 equivalent) and Xantphos
To a round-bottom flask containing DMF (1.00 mL, 12.98 mmol, 1.3
equivalents, Supelco®) kept in an ice bath under a nitrogen atmosphere,
phosphoryl chloride (POCl , 1.21 mL, 12.98 mmol, 99% purity, 1.3
(11.93 mg, 0.021 mmol, 0.05 equivalents) were added. The system was
purged with nitrogen for 15 minutes. Afterwards, degassed triethyl-
amine (99% purity, 3.30 mL, 0.13 M, Sigma-Aldrich, USA) was added,
3
equivalents, Sigma-Aldrich, USA) was slowly added using a syringe.
After the addition, the color of the reaction became light pink. After that,
and 3-butyn-1-ol (40.6 L, 0.54 mmol, 97% purity, 1.3 equivalents,
μ
Sigma-Aldrich, USA) was added to the obtained suspension. The reac-
tion stirred for 3 hours. The reaction mixture was diluted with ethyl
acetate (15 mL) and washed with 1 M HCl (2 × 15 mL), distilled water
(2 × 10 mL) and brine (20 mL). Afterwards, the organic layer was dried
over anhydrous sodium sulfate, evaporated under reduced pressure and
′
a solution of 2,2 -bithiophene in DMF (5 mL) was added into the pre-
viously prepared pink solution. The cooling bath was removed, and the
◦
reaction was heated to 80 C and stirred until complete consumption of
◦
the starting material. After that, the reaction was cooled to 0 C, and a
cold solution of saturated sodium acetate (NaOAc, 15 mL) was added.
This solution was vigorously stirred for 3 hours, and after that time, the
reaction mixture was diluted with diethyl ether (30 mL) and washed
with sodium hydroxide (1 M NaOH, 15 mL), water (15 mL) and brine (15
mL). The organic layer was dried over anhydrous sodium sulfate,
2
purified by flash column chromatography [SiO , hexane:ethyl acetate
(70:30, v/v)] to provide 2c as a light yellow solid (66.9 mg, 62% yield),
1
which will be referred to as Thio2. H NMR (400 MHz, DMSO-d6) δ 7.49
(dd, J = 5.2, 1.0 Hz, 1H), 7.21 (dd, J = 3.6, 1.1 Hz, 1H), 7.03 (dd, J =
5.2, 3.6 Hz, 1H), 4.56 (s, 1H), 3.59 (t, J = 6.8 Hz, 2H), 2.59 (t, J = 6.8
Hz, 2H).
evaporated and subjected to flash column chromatography (SiO
2
, hex-
ane) to provide light yellow needles (1.12 g, 58% yield) as the final
1
′
product. H NMR (250 MHz, CDCl
3
) δ 9.86 (s, 1H), 7.66 (d, J = 3.9 Hz,
2.6. Experimental procedure for the synthesis of 4-(5 -(hydroxymethyl)-
′
1
H), 7.42-7.32 (m, 2H), 7.25 (d, J = 3.9 Hz, 1H), 7.13-6.99 (m, 1H).
[2,2 bithiophene]-5-yl)but-3-in-1-ol
′ ′
.4. Experimental procedure for the synthesis of 5 -iodo-[2,2 -
2
bithiophene]-5-carbaldehyde
4
Sodium borohydride (NaBH ) (34.61 mg, 0.91 mmol, 99%, 4.0
equivalents, Sigma-Aldrich, USA) was added to a stirred solution of 2c
◦
(
60.0 mg, 0.23 mmol, 1.0 equivalent) in absolute ethanol at 0 C (2.30
mL, 0.4 M). The reaction continued until all the starting material was
consumed. The solvent was evaporated under reduced pressure, and the
crude reaction product was diluted with ethyl acetate (30 mL) and
4
washed with saturated NH Cl solution (15 mL), water (15 mL) and brine
The p-toluene sulfonic acid (TsOH) [44.32 mg, 0.26 mmol, 98%
(15 mL). The organic layer was dried over anhydrous sodium sulfate,
evaporated and subjected to purification by flash column chromatog-
purity, 0.1 equivalent, Sigma-Aldrich, USA] was added to a solution of
2
a (500.0 mg, 2.57 mmol, 1.0 equivalent) in chloroform/acetic acid
raphy [SiO
2
, hexane:ethyl acetate (70:30, v/v)] to provide product 2d as
◦
1
(
1:1, 15 mL, 0.17 M) and cooled to 0 C, and the reaction mixture stirred
an amorphous yellow solid (58.0 mg, 96% yield). H NMR (250 MHz,
3

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
MeOD-d4) δ 7.05 (d, J = 3.7 Hz, 1H), 7.01 (d, J = 4.1 Hz, 2H), 6.90 (d, J
nonencapsulated compound and obtain nonaggregated nanoparticles.
The particle size and the polydispersity index (PDI) of the prepared
piperine nanoparticles were characterized using dynamic light scat-
tering (DLS) with a submicron particle size analyzer (Zetasizer Nano-
ZS™, Malvern Panalytical Instruments, UK), equipped with 4.0 mV He-
=
3.6 Hz, 1H), 4.70 (s, 2H), 3.72 (t, J = 6.7 Hz, 2H), 2.65 (t, J = 6.7 Hz,
13
2
H) C NMR (63 MHz, MeOD-d4) δ 146.12, 139.17, 137.49, 133.31,
1
26.83, 124.72, 124.12, 123.76, 93.30, 75.31, 61.49, 60.01, 24.52.
′
Compound 2d, a slight modification of 5-(4-hydroxybut-1-inyl)-2,2 -
◦
bithiophene previously identified in a crude ethanolic extract from the
Ne laser (λ = 633 nm), applying 175 detection angle to DTS1070
aerial parts of T. patula will be referred to as Thio1.
capillary cuvettes. The zeta potential was characterized using the same
instrument with a zeta potential analyzer by determining the electro-
phoretic mobility of nanoparticles under an electrical field. All mea-
surements were performed in triplicate.
2
2
.7. In vitro bioassays
.7.1. Egg hatch test (EHT)
The egg hatch test was performed with the samples of Thio1 and
2.9. In vivo bioassay
Thio2 in order to choose the most appropriate compound for the prep-
aration of the nanostructured formulation to be administered in the in
vivo test. The faeces were directly collected from the rectal ampoule of
the Santa In ˆe s sheep and later processed according to the technique of
Coles et al. (1992) modified by Bizimenyera et al. (2006). Approxi-
mately 100 eggs per volume of H. contortus were placed in each well of a
2.9.1. Fecal egg count reduction test (FECRT)
Santa In
ˆe s sheep aged between 8 and 9 months were used (Embrapa
Pecu
a´ ria Sudeste Ethics Committee on Animal Experimentation, process
n. 04/2017). For artificial infection, the animals received oral treatment
with the organophosphate trichlorfon (Neguvon®, Bayer, Germany), 1
ꢀ 1
2
4-well cell culture plate and the volume was completed to 250 µL using
mL.kg from a 10% (v/v) solution. After three consecutive measure-
ments with zero egg counts in the fecal mass of the sheep, each animal
distilled water. Then, 250 µL of the each test samples were added,
obtaining final concentrations ranging from 4 to 0.001 mg.mL (15 to
ꢀ 1
3
was inoculated with approximately 4,000 larvae (L ) of H. contortus
ꢀ 3
3
.75 × 10 mM). The negative control consisted of distilled water and
from the Embrapa 2010 isolate, characterized as resistant to benzimid-
azoles, imidazothiazoles and macrocyclic lactones (Chagas et al. 2013).
After 28 days of artificial infection, the sheep were weighed, and their
fecal matter was collected directly from the rectum of the animals in
order to proceed to the egg count per gram of faeces [EPG] (Ueno and
Gonçalves 1998).
Tween 80 2% v/v (Sigma-Aldrich, USA), and a positive control with
ꢀ 1
thiabendazole (Sigma-Aldrich, USA) at 60 g.mL was used (Gainza
μ
et al., 2015; Gainza et al., 2016). The test was performed with six rep-
etitions. The plates were homogenized manually and stored in a
◦
Biochemical Oxygen Demand incubator BOD (27 C and 80% RH) for 24
hours. The results were measured by adding a drop of lugol to each well,
The animals were distributed through three test groups with n = 10,
constructed in order to be homogeneous in relation to the initial para-
sitic load and weight: (i) negative control (untreated); (ii) positive
control (Zolvix™ (monepantel), Elanco, USA); (iii) nano Thio1 group.
Determination of the doses to be orally administered were calculated
according to the weight of the animals (2.5 mg of each product/kg of live
weight). During the period of the experiment, the animals were kept in
housed in individual metabolic cages (2.68 × 0.80 × 2.02 m, L x W x H),
receiving controlled feed and water ad libitum. The animals had daily
veterinary follow-ups to observe any adverse reactions to the products
administered, as well as other signs that pointed to a disorder in its vital
conditions (anemia by Famacha scoring system, body temperature,
diarrhea, foam in the mouth, etc). The EPG count was performed for all
1
and the eggs and hatched L were counted. These data were used to
calculate the percent hatchability inhibition, according to the equation
below:
(
Num ber of eggs)
EHT =
x 100
(
Num ber of eggs + Num ber of L1)
2
.7.2. Larval development test (LDT)
Approximately 100 eggs recovered from sheep faeces were placed in
each well of a 24-well plate together with 10 µL of amphotericin B
ꢀ 1
(
0.125 mg.mL stock solution) and 20 µL of nutrient medium, diluting
to a volume of 250 µL with distilled water (Hubert and Kerboeuf 1992).
◦
The plates were incubated (27 C and 80% RH), and after 24 hours, 250
2
groups on day 0 (EPG ) and after 3, 7, 10, 14, 17, 21, 24 and 29 days. In
µL of the test solutions were added to L
1
, obtaining concentrations from
order to check whether the formulation had any effect on the hatch-
ability of eggs, fecal samples from animals in the negative control group
and in the group treated with nano Thio1 were collected 2, 9, 16, 23 and
ꢀ 1
ꢀ 3
1
0.0 to 0.002 mg.mL (37 to 7.57 × 10 mM). The negative control
consisted of
a dimethyl sulfoxide solution 0.5% v/v (DMSO,
Sigma-Aldrich, USA) and distilled water, and a positive control with
3
0 days after treatment to perform the EHT. The calculation of the ef-
ꢀ 1
ivermectin (Sigma-Aldrich, USA) at 0.01 mg.mL was used (Gainza
et al., 2015; Gainza et al., 2016; Politi et al., 2018). The test was per-
ficacy of nano Thio1 was carried out using the following equation
(
Wursthorn and Martin, 1990):
formed in six repetitions. After incubation for 6 days, live L
1
+ L
2 3
and L
(
%)Efficacy(Sample) = EPG
Control ꢀ EPGSample
x 100
larvae were counted to estimate the percent inhibition of larval devel-
opment, according to the equation:
EPGControl
(
L1 + L2)
LDT =
x 100
2.10. Molecular modeling studies
(
Total num ber of larvae)
The H. contortus GluCl (UniProt ID P91730) primary structure was
submitted to the BLASTP server (blast.ncbi.nlm.nih.gov) to search against
the Protein Data Bank (PDB) for the crystallographic structure with the
highest sequential identity and sequence cover. Caenorhabditis elegans
GluCl cocrystallized with ivermectin (PDB ID: 3RHW) was selected
(Hibbs and Gouaux 2011). The Chains A and B located in the trans-
membrane domain were prepared for molecular modeling studies using
the Protein Preparation tool available on the Maestro v.10.8 interface
(Schr o¨ dinger, USA). Solvent molecules and the cocrystallized ligands
were removed, and hydrogen atoms were added to the protein model.
The protonation state for histidine, glutamine and asparagine residues at
pH 7 ± 2 was optimized, as well as the hydrogen bonds. Energy mini-
mization was performed using the OPLS2003 force field. Nonconserved
2
.8. Preparation and characterization of polycaprolactone nanoparticles
containing Thio1
The polymeric nanoparticles (nano Thio1) were prepared by the
nanoprecipitation method (Fessi et al. 1999). Briefly, the organic phase
(
OP) containing thiophene (Thio1, 375 mg), polycaprolactone (PCL,
875 mg, Sigma Aldrich, USA) and acetone (50 mL) was slowly dripped
into the aqueous phase (AP) containing 0.5% surfactant (poloxamer
07, 100 mL, Sigma-Aldrich, USA) under magnetic stirring (1000 rpm)
in an exhaust hood until complete solvent evaporation. The resulting
suspension was subjected to filtration (0.45 m membranes), followed
by ultracentrifugation (40,000 rpm), in order to eliminate the
1
4
μ
4

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
residues of the ivermectin binding site in H. contortus were mutated and
minimized using the Prime v.10.8 program (Jacobson et al. 2002,
Jacobson et al. 2004, Schr o¨ dinger 2016). After mutation, the Receptor
Grid Generation tool was used to create a cubic grid with 10 Å edges
centered on the residues T257, Q260 and T285 located in chain A, in
addition to the residue V218 from chain B (Halgren et al. 2004, Friesner
et al. 2004, Friesner et al. 2006). Moreover, the 3D structure of the
thiophene derivative was submitted to the Ligand Preparation tool to
compute charges (OPLS 2003 force field) and structural optimization.
All resulting conformers were submitted to docking, first using a stan-
dard precision module (SP), followed by docking in the extra precision
module (XP). During the conformation sampling step, the ligand struc-
tures were considered flexible. No restrictions on the interaction with
ivermectin binding site residues were applied, aiming to explore the
binding site pocket and the conformations of the ligands. The poses were
ranked using the GlideScore function and visually inspected to evaluate
the binding mode and interactions between ligands and GluCl binding
site residues (Friesner et al. 2004).
material and the mono- and dibrominated products have very close
retention coefficients on silica, making it difficult to separate the prod-
uct of interest from the reactional medium. To minimize this inconve-
nience,
a
small
experimental
modification
was
made:
N-bromo-succinimide (recrystallized just before being used on boiling
water) was added in small portions to prevent 1a from reacting with
itself after it formed. However, some of the dibrominated product was
still observed just above the product of interest on the chromatographic
2
plate (SiO , hexane). In order to achieve an efficient separation, a 100:1
silica/crude mass ratio was used, making it possible to obtain the
product of interest in reasonable yield (59%). After obtaining 1a, a
Sonogashira coupling was performed with 3-butyn-1-ol. The Sonoga-
shira coupling is an extremely robust reaction catalyzed by palladium,
sometimes together with copper, which allows a coupling between ter-
minal alkynes and chlorinated, brominated, or iodinated compounds,
with the latter two being more reactive (Chinchilla and N a´ jera 2011,
Shilz and Plenio 2012). Thus, 1a was placed in the presence of catalytic
palladium acetate and catalytic copper iodide with cesium carbonate as
the base and using a bisphosphine as a binder, in order to minimize the
formation of “black palladium”. The reaction satisfactorily generated 1b
in excellent yield (91%) and with simple purification (the products
separated well on the chromatographic plate).
2
.11. Statistical analysis
The obtained results from the in vitro tests were subjected to analysis
of variance (ANOVA), with the means being compared by the parametric
test of Tukey (p ≤ 0.05), using Minitab® 19 software. For the calculation
of effective concentrations (EC50 and EC90), probit analyses were per-
formed using Minitab® 19. The degree of data adjustment was assessed
by Pearson’s chi-square test using the maximum likelihood estimation.
To obtain derivative 2e, which has a hydroxyl on the side chain also
′
starting from 2,2 -bithiophene, a Vilsmeier-Haack formylation reaction
was performed (Su et al. 2010). The choice to formylate the thiophene
was because the addition of a carbonyl to the ring makes deactivates it
for aromatic electrophilic substitution reactions, as well as the occur-
rence of problems with a second functionalization just as what happened
during the bromination to obtain 1a (Raimundo et al. 2001, Cui et al.
As for the in vivo assay, the EPG values were transformed using log10
,
and the resulting data were subjected to analysis of variance (ANOVA)
with the means being compared by either Tukey’s or Fisher’s LSD test
3
2011). As predicted, after the reaction using POCl in the presence of
(
Minitab® 19).
anhydrous DMF, monoformylated product 2a was selectively obtained
and easily purified by a chromatographic column in quantitative yield
after recovery of the starting material. As 2a already has a substituent at
3
. Results and discussion
′
position 5 of 2,2 -bithiophene, the iodination or bromination reaction
3
.1. Synthesis of the compounds
provides a single product 2b that is easily recrystallized using heated
ethanol. The iodized product 2b was subjected to the Sonogashira
coupling reaction to provide 2c, which was subjected to reduction with
sodium hydride and boron in ethanol to provide 2d.
To obtain some of the thiophenes previously identified in the etha-
nolic extract of T. patula (Politi et al. 2018), three synthetic routes were
′
proposed starting from 2,2 -bithiophene (Figure 1). Several precedents
can be found in the literature with methodologies for the mono-
3.2. In vitro bioassays
′
bromination of 2,2 -bithiophene (Zhao et al. 2010, Krompiec et al.
ꢀ
1
2
015); nevertheless, when testing several of them, a mixture of products
For EHT, the initial concentration for both samples was 4 mg.mL
(15 mM). Thio1 showed 100% efficacy in inhibiting egg hatching at a
containing 1a, the dibrominated product, was obtained in addition to
unconsumed starting material. The problem of using a methodology that
is not selective in this first stage was found to be that both the starting
ꢀ 1
concentration of 1 mg.mL (3.7 mM) [Table 1], with an EC50 = 0.3243
ꢀ 1
ꢀ 1
mg.mL (1.2 mM) [95% CI: 0.3035 – 0.3464 mg.mL ] and EC90
=
ꢀ 1
0
.8228 (3.1 mM) [95% CI: 0.7769 – 0.8751 mg.mL ]. This EC50 value is
approximately 40 times lower than the value found for the crude etha-
ꢀ 1
nolic extract of T. patula [EC50 TpEtOH = 12.8 mg.mL (48 mM)] (Politi
ꢀ 1
et al., 2018). At concentrations below 0.01 mg.mL (0.037 mM), the
inhibition values were not significantly different from those presented
by the negative controls at the 5% significance level (Table 1). For
comparison, Lone et al. (2012) using the commercial nematicide le-
ꢀ 1
vamisole found an EC50 value = 1.88 mg.mL (9.2 mM), which is
approximately 7.6 times higher than that of Thio1. In another study,
Ribeiro et al. (2014) using the nanoencapsulated essential oil of Euca-
ꢀ 1
lyptus citriodora found an EC50 = 0.40 mg.mL . Thio2 showed no
relevant anthelmintic activity under the assay conditions (EC50 > 4 mg.
ꢀ 1
mL ) and did not differ statistically from the negative controls at 6 of
the 7 concentrations applied (Table 1). The thiabendazole used as a
positive control showed 100% efficacy in the applied concentration
ꢀ
1
(
0.06 mg.mL ).
For the LDT, due to the low efficacy of Thio2 observed in the EHT,
ꢀ 1
only Thio1 was tested, starting at 10 mg.mL (37 mM). The compound
showed 100% inhibition of larval development down to the concentra-
ꢀ 1
tion of 0.625 mg.mL (2.3 mM) [Table 2], with an EC50 = 0.1731 mg.
ꢀ 1
ꢀ 1
Figure 1. Synthetic route used in the synthesis of selected thiophenes.
mL (0.65 mM) [95% CI: 0.1676 – 0.1791 mg.mL ] and EC90 =
5

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
Table 1
Thio1 and Thio2 against Haemonchus contortus on egg hatch test (EHT).
ꢀ
1
§
ꢀ 1
§
Sample
Thio1
C (mg.mL
)
I (%) (± SD)
Sample
Thio2
C (mg.mL
)
I (%) (± SD)
a
a
1.0
100 (± 0)
4.0
22.1 (± 10.6)
b
a,b
0
0
0
0
0
0
.8
77.2 (± 3.4)
2.0
18.1 (± 7.6)
c
a,b
.4
63.4 (± 5.3)
1.0
15.0 (± 2.4)
d
a,b
.2
47.4 (± 0.5)
0.5
13.6 (± 4.4)
d
a,b
.1
37.4 (± 6.4)
0.25
0.12
0.06
0,06
13.4 (± 2.1)
d
a,b
.05
.025
36.2 (± 11.8)
11.7 (± 6.2)
e
a,b
22.3 (± 0.1)
10.6 (± 2.5)
a
a
Thiabendazole
0,06
100 (± 0)
Thiabendazole
100 (± 0)
f
f
Negative Control
Distilled water
Tween 80 2%
9.0 (± 3.0)
Negative Control
Distilled water
Tween 80 2%
9.0 (± 3.0)
f
f
7.5 (± 1.2)
7.5 (± 1.2)
§
I (%) = percentage of inhibition. Means with different superscript letters in the same column exhibit statistically significant differences by Tukey Test (p ≤ 0.05).
ꢀ
ꢀ 1
0
.2646 mg.mL 1 (1.0 mM) [95% CI: 0.2551 – 0.2752 mg.mL ]. This
tendency to permeate membranes. Besides, cationic nanoparticles cause
more pronounced disruption of plasma-membrane integrity, stronger
mitochondrial and lysosomal damage, and a higher number of auto-
phagosomes than anionic nanoparticles (Frohlich, 2012), limiting its use
due to greater cytotoxicity. The zeta potential values around +30 mV or
-30 mV represents electrokinetic stability of the formulation, indicating
that the thickness of the electrical double layer is maintained, prevent-
ing aggregation due to the strong repulsive forces between the particles
EC50 value is approximately half the value found for the crude ethanolic
ꢀ 1
extract of T. patula [EC50 = 0.34 mg.mL ] (Politi et al. 2018) and
approximately half the value found for the hydroalcoholic extract of
ꢀ 1
grape marc [EC50 = 0.30 mg.mL ] (Soares et al. 2018). The ivermectin
used as a positive control showed 100% efficacy in the applied con-
ꢀ 1
centration (0.01 mg.mL ).
(
(
Bhattacharjee 2016). Regarding the choice of surfactant, Dunn et al.
1997) compared polymeric nanoparticles coated with poloxamers 407
3
.3. Preparation and characterization of polycaprolactone nanoparticles
containing Thio1
and 188, and, through in vivo studies, demonstrated that while nano-
particles coated with poloxamer 188 were readily metabolized with
rapid clearance in the liver and spleen, nanoparticles coated with
poloxamer 407 showed prolonged action in the bloodstream, optimizing
its controlled release and justifying its use in the present work.
Biodegradable polymers have been one of the main focuses of at-
tempts to develop improved controlled release systems for pharmaceu-
tical research. The most common are aliphatic polyesters and their
copolymers (Thombre and Cardinal 1990). Polycaprolactone (PCL) is a
synthetic aliphatic polyester approved by the FDA and has some ad-
vantages: it is hydrophobic, biodegradable, biocompatible and relatively
inexpensive (Sinha et al. 2004). In addition, due to its low toxicity, it is
suitable for intravenous or oral administration (Chen et al. 2000, De La
Ossa et al. 2012, Tian et al. 2012). In this work, polymeric nanoparticles
carrying Thio1 were prepared by the nanoprecipitation or interfacial
deposition technique (Fessi et al. 1989), which is an effective, economic,
easy to perform, and scalable technique that requires the use of less
organic solvent and consists of the dripping of an organic phase (con-
taining a volatile water-miscible solvent, the drug and a polymer) in an
aqueous solution with a surfactant under constant agitation. The poly-
mer, once in contact with the aqueous solution, precipitates together
with the drug forming the nanoparticles (Legrand et al. 2007, Hirsjarvi
et al. 2008). The particle size of the prepared nanoparticles measured by
DLS was 228.30 ± 2.77 nm with a PDI value of 0.165 ± 0.015 (Figure 2).
With such size and monodisperse nature, the prepared nanoparticles
were suggested to be suitable for further in vivo anthelmintic assay, since
the small size of the particles facilitates the absorption of the compound
and increases its bioavailability. In addition, the nano Thio1 showed a
zeta potential of -29.80 ± 0.208 mV. Since most cellular membranes are
negatively charged, a negative zeta potential can affect a nanoparticle’s
3.4. In vivo bioassay
Based on the outstanding Thio1 in vitro activity, an in vivo assay using
an animal matrix as a proof of concept was performed. Two strategies to
evaluate the in vivo activity were investigated: (i) direct action by
reducing the number of eggs in the faeces of animals (EPG count over a
month) and (ii) indirect action by a decrease in the hatching of eggs in
the faeces of animals (periodic count of hatched eggs over a month by
the EHT). Table 3 shows the values found for the EHT counts after
treatment with nano Thio1. The formulation showed low inhibitory
activity on day 2 posttreatment. On day 9, the percent inhibition did not
significantly differ from the control. It is logical to state that the meta-
bolic stability of the product is low; however, the short exposure time to
the animal was sufficient to cause a slight decrease in the hatching of the
eggs. It is worth mentioning that the eggs are released close to the faeces
of the sheep, there was low inhibitory action by the orally administered
1
nanoformulation, and a smaller amount of L larvae would be free in the
surroundings where the animals graze. The interactions between the
nanoparticles of the formulation and the cells of the sheep intestinal
mucosa are quite complex, and specific studies must be carried out in
Table 2
Thio1 against Haemonchus contortus on larval development test (LDT).
ꢀ
1
Sample
Thio1
C (mg.mL
)
L
1
+ L
2
L
3
Total
(%) Inhibition ± SD
a
0.625
104.66
104.83
34.00
14.83
2.66
0
104.66
105.50
105.83
107.00
103.16
105.16
107.33
108.16
105.33
100 (± 0)
a
0.312
0.156
0.078
0.039
0.0019
0.66
99.37 (± 0.97)
b
71.83
92.16
100.50
104.16
0
32.12 (± 0.68)
c
13.87 (± 1.02)
d
2.58 (± 0.50)
d
1.00
0.93 (± 1.02)
a
Ivermectin
0.01
107.33
1.33
100 (± 0)
d
Negative control
Distilled water
DMSO 0.5%
106.83
103.83
1.22 (± 0.45)
d
1.50
1.41 (± 0.75)
Means with different superscript letters in the same column exhibit statistically significant differences by Tukey Test (p ≤ 0.05).
6

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
Figure 2. Graphical representation of the nanoformulation size distribution profile by DLS method. (A) Size distribution curve for control nanoparticles (blank); (B)
Size distribution curve for Thio1 loaded nanoparticles.
Table 3
Periodic monitoring of eggs recovered from animals of the negative control group (C-) and animals treated with the nanoformulation (nano Thio1).
Day
Egg Hatch Test (EHT)
◦
◦
Eggs + N◦
N
Eggs
N
L
1
(%) Inhibition (± SD)
C-
Nano Thio1
13,50
6,67
C-
Nano Thio1
103,33
82,50
C-
Nano Thio1
a
b
Day 2
6.67
5.67
7.92
7.25
3.00
114.83
87.83
5.76 (± 1.55)
12.80 (± 5.42)
a
a
Day 9
6.60 (± 1.53)
7.97 (± 4.22)
a
a
Day 16
Day 23
Day 30
10,67
8,33
133.25
127.08
73.33
166,25
146,00
95,33
6.00 (± 2.49)
6.40 (± 1.87)
a
a
5.68 (± 1.68)
5.77 (± 1.59)
a
a
4,42
4.08 (± 1.83)
4.71 (± 1.60)
Means with different superscript letters in the same column exhibit statistically significant differences by Tukey Test (p ≤ 0.05).
order to optimize this inhibitory activity, seeking to provide a greater
dose of the compound at the site of action before its metabolism,
maximizing its intact passage through the ruminal compartment.
Table 4 shows the average EPG values for the three tested groups over
one month. As can be observed, there is a regularity in the values found
in all the groups, allowing some inferences: (i) in the untreated group
nematodes, more specifically in the subunits Cel-acr-23 (or ACR-23) and
Hco-mptl-1 [MPTL-1] (Spinosa et al. 2014). Although quite efficient, the
cost of the product is relatively high (250-300 USD per liter); further-
more, because of indiscriminate and inappropriate use, there are risks of
a rapid generation of resistant nematode strains. In this sense, Scott
et al. (2013) evaluated the effectiveness of monepantel in goats and
sheep using the egg reduction test and observed a failure in efficacy
against Capillaria spp., Oesophagostomum venulosum, Strongyloides spp.,
Teladorsagia circumcincta, Trichostrongylus colubriformis and Trichuris spp.
Mederos et al. (2014) also reported the inefficiency of monepantel to
control the multidrug-resistant strain of H. contortus in sheep flocks from
two different farms in Uruguay; (iii) in the group using nano Thio1
(
negative control), the EPG values varied around an average in the in-
termediate readings (EPGmean = 3116.6 between days 3 and 10 and
EPGmean = 3316.6 between days 17 and 24); however, the middle (EPG
=
4207.1 on day 14) and end of the experiment (EPG = 4842.8 on day
3
0) presented indexes that represent slightly more than twice the initial
reading (Figure 3, Table 4). In general, it can be assumed that the in-
creases in these two distinct periods refer to the normal parasite cycle of
infestation in the host. The eggs start to be eliminated at approximately
′
′
[4-(5 -(hydroxymethyl)-[2,2 -bithiophene]-5-yl)but-3-yn-1-ol
nano-
encapsulated], the EPG values were maintained, on average, close to the
initial count value (EPGmean = 2167.1 against EPGinitial = 2425.0).
Although there was no marked drop in parasitic load as observed in the
group treated with the vermifuge, there was also no significant increase
as was observed in the negative control group (Figure 3, Table 4).
Interestingly, the administration of the product still in its preliminary
form resulted in the maintenance of a stable load of eggs in the hosts,
showing a mean efficacy of 36.45%. It should be mentioned that EPG
values up to 3,000 are not as impactful for sheep, especially for hair
sheep such as Santa In ˆe s and Morada Nova, because they are more
resistant and subjected to deworming only when they reach an EPG of 4,
2
1 and 28 days and tend to increase until day 90. Thus, the initial load of
eggs that was eliminated with the faeces in the period close to two weeks
after the beginning of the experiment was possibly responsible for the
reinfestation of the sheep, culminating in a very high count at the end of
4
weeks; (ii) in the Zolvix™ group (positive control), the EPG values of
all animals had already declined dramatically from the first collection of
results after treatment, remaining low throughout the experiment
(
Figure 3, Table 4). Zolvix™ (monepantel) is a recently developed
anthelmintic from the class of amino acetonitrile derivatives (AADs) and
acts on the nicotinic receptor (nAchR), which is found only in
Table 4
Mean values of egg count per gram of feces (EPG) and weight (Wt) for animals submitted to fecal egg count reduction test (FECRT).
Day
C-
Zolvix™ (monepantel)
EPG
Nano Thio1
EPG
EPG
Wt (kg)
Wt (kg)
Wt (kg)
a
a
a
a,b
a
a
0
3
7
2178.6
4242.8
2857.1
2250.0
4207.1
3021.4
3592.8
3335.7
4842.8
35.6
2321.4
34.4
2425.0
23.5
a,b
a,b
a
b
a
-
28.5
-
1750.0
-
a
b
b
a
a
35.5
21.4
33.9
2025.0
22.1
b
a
10
14
17
21
24
30
-
7.14
-
1812.5
-
a,b
a,b
a,b
a,b
b
a
b
a,b
a
a
37.5
7.14
36.7
2400.0
24.5
b
a
-
7.14
-
2037.5
-
a
b
a,b
a
a
37.8
14.29
36.7
2150.0
24.2
b
a
-
14.29
-
2612.5
-
a
b
a
a
a
38.3
7.14
37.6
2550.0
24.4
Means with different superscript letters in the same column exhibit statistically significant differences by Tukey Test (p ≤ 0.05).
7

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
light on the mode of action of thiophenic derivatives, molecular
modeling studies were carried out on the glutamate-activated chloride
channel (GluCl), a well-known anthelmintic molecular target. In mam-
mals, the Cys-loop superfamily can be broadly categorized between
nicotinic and serotonin excitatory receptors (nAChR and 5-HT3,
respectively) and inhibitory receptors [GABA or glycine-dependent
chloride channels] (Miller and Smart 2010). In the nematode
C. elegans, inhibitory neurotransmission is mediated, in part, by GABA
ionotropic receptors (UNC-49 receptors), which are concentrated in the
neuromuscular junctions where they play a fundamental role in loco-
motion and excretion (Bamber et al. 2003, Gally et al. 2003, Schuske
et al. 2004). The unc-49 gene encodes proteins for three subunits of the
GABA receptor (UNC-49A, UNC-49B and UNC-49C); however, only
UNC-49B and UNC-49C are expressed at physiologically relevant levels
and join to form the native receptor in vivo (Bamber et al. 1999, Bamber
et al. 2005). While UNC-49C has modulatory action and does not seem to
be essential for receptor function in vivo, its association with UNC-49B
causes a decrease in sensitivity to both GABA and channel blocking
picrotoxins in addition to directly contributing to the sensitivity to
neurosteroids (Bamber et al. 2003, Wardell et al. 2006). The UNC-49
channel is also present in H. contortus (Hco-UNC-49); however, it is
2
.5 times more sensitive to GABA than the homologous channel in
C. elegans (Wardell et al. 2006, Siddiqui et al. 2010). The structural
conservation of the transmembrane domain among species supports the
selection of GluCl from C. elegans in complex with ivermectin as the
template for modeling (Hibbs and Gouaux 2011). Moreover, high
sequential identity (57%) and structural conservation between GluCl of
the nematodes H. contortus (UniProt ID P91730) and C. elegans (PDB ID
Figure 3. Results of the in vivo anthelmintic test. (A) Mean EPG values for
treatments using monepantel (Zolvix™), Thio1 nanoencapsulated formulation
3
RHW) is observed (Figure 5). The selected crystallographic model was
(
nano Thio1) and negative control; (B) Logarithmic trend lines of the obtained
EPG results.
properly prepared for docking studies based on mutation and minimi-
zation of nonconserved residues to mimic the H. contortus GluCl active
site. Then, the 3D thiophene derivative conformers were docked into
H. contortus GluCl at the ivermectin binding site. Docking methods use
energy functions including bonded and nonbonded parameters (e.g.,
hydrogen bonds, electrostatics, van der Waals, and hydrophobic in-
teractions) to generate mathematical models that predict the most
favorable ligand orientations according to a sorted list of energy scores
0
00 (Chagas et al. 2016, Toscano et al. 2019). Thus, the administration
of nano Thio1 conferred natural control that can be used in combination
with the usual chemical treatment. It is important to mention that the in
vivo test was a complement to in vitro assays, having been carried out in a
preliminary way just to have an idea about the possibility of orally
administering a nanoformulation in the tested animals, observing
whether there would be adverse reactions or intoxication signs. The
perspectives reported here are good and encourage further in-depth
studies.
(
Alonso et al. 2006) (Figure 4).
In this work, all conformations were ranked using the GlideScore
3
.5. Molecular modeling studies
The rational design based on structure and mechanism of action
constitutes a very attractive strategy for the discovery of new drug
candidates. This strategy has the potential to contribute at all stages of
the process, initiating with the discovery of prototypes or starting
compounds from isolation in plant species followed by optimization
until elaboration of formulations that are ready for clinical tests (Man-
dal et al. 2009). This approach is based on blocking or stimulating the
biological activity of macromolecules associated with different meta-
bolic processes, such as those associated with pathogenicity, detoxifi-
cation, and locomotion mechanisms, among others. The structural
information of the ligands within catalytic sites allows the discovery and
synthesis of compounds with steric, hydrophobic and/or electrostatic
complementarity. The convergence of genomic technologies and the
discovery of new drugs against specific molecular targets enables the use
of bioinformatics tools, optimizing the transition between biological
knowledge and clinical therapy. This can be achieved, for instance, by
identifying genes that have properties similar to the targets already
described, investigating pairwise similarity between genomic libraries
from different origins, such as those of susceptible or pesticide-resistant
strains, and building models of receptor targets based on sequential
homology and structural similarity (Desany and Zhang 2004). To shed
Figure 4. Thiophene derivative 1 (Thio1) pose (sticks) obtained by docking
into the ivermectin binding site of the H. contortus GluCl model (gray surface).
Residues interacting by hydrogen bonds with the ligand of interest are repre-
sented in lines, as well as the residues at positions 257, 260, 285 and 218, which
interact with ivermectin. These residues are in the chain A, apart from the
residue 218 located in the chain B. Hydrogen bonds are indicated as black
dashes. The Thio1 and the main interacting residues are represented with
carbon in white and cyan, respectively, oxygen in red, nitrogen in blue, sulfur in
yellow and polar hydrogens in white. For clarity, non-polar hydrogens atoms
were omitted. Figure generated using PyMol (v 1.7.2.1).
8

F.A.S. Politi et al.
Acta Tropica 219 (2021) 105920
Figure 5. Sequence alignment of the GluCl
from H. contortus (Uniprot ID P91730) and
C. elegans (PDB ID 3RHW). Residues are colored
in blue according to the sequence identity, with
identical residues represented in deep blue. The
residues interacting by hydrogen bonds with
the thiophene derivative are highlighted, with
the C. elegans residue numbering scheme indi-
cated above the highlighted residue as refer-
ence. Figure generated using Jalview v.2.11.0,
with alignment carried out by T-Coffee.
function, which simulates binding free energy; therefore, higher nega-
tive values indicate ligands with higher affinity for the molecular target
Alonso, H, Bliznyuk, AA, Gready, JE., 2006. Combining docking and molecular dynamic
simulations in drug design. Med Res Rev 26 (5), 531–568.
Bamber, BA, Beg, AA, Twyman, RE, Jorgensen, EM., 1999. The Caenorhabditis elegans
unc-49 locus encodes multiple subunits of a heteromultimeric GABA receptor.
J Neurosci 19 (13), 5348–5359.
(
Halgren et al. 2004, Friesner et al. 2004, Friesner et al., 2006). The best
ꢀ
1
ranked Thio1 pose showed a GlideScore value of -5.7 kcal.mol . The
predicted binding mode of Thio1 within the H. contortus GluCl model
indicates that the compound fits into the ivermectin binding site. Iver-
Bamber, BA, Twyman, RE, Jorgensen, EM., 2003. Pharmacological characterization of
the homomeric and heteromeric UNC-49 GABA receptors in C. elegans. Br J
Pharmacol 138 (5), 883–893.
ꢀ 1
mectin, with a molecular weight of 875 g.mol , allosterically binds in
the C. elegans GluCl transmembrane domain, establishing an extensive
network of hydrophobic interactions and three hydrogen bonds with
residues S260, T285 and L218 (Hibbs and Gouaux 2011). Thio1 (MW=
Bamber, BA, Richmond, JE, Otto, JF, Jorgensen, EM., 2005. The composition of the
GABA receptor at the Caenorhabditis elegans neuromuscular junction. Br J Pharmacol
1
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64.03 g.mol ) establishes two hydrogen bonds with active site resi-
dues, one with the side chain of T285 and the other with the main chain
carbonyl group of D277. Therefore, based on the affinity score and
proposed binding mode, Thio1 is a potential candidate ligand of the
glutamate-activated chloride channel.
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5
. Conclusions
2
In conclusion, Thio1 demonstrated high anthelmintic action in the in
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vitro assays, showing activity superior to that found in other studies.
Regarding the in vivo test, the preliminary results encourage new studies
in which adjustments and refinements will be performed in the prepa-
ration of the nanoformulation in order to better understand its phar-
macokinetics and stability, thereby increasing its biological activity.
Moreover, starting from Thio1, modifications and/or insertion of other
functional groups can be designed to discover and develop more potent
compounds.
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biomaterials - Poly (epsilon-caprolactone). Sheng Wu Yi Xue Gong Cheng Xue Za Zhi
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World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.)
methods for the detection of anthelmintic resistance in nematodes of veterinary
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Author statement
′
Cui, M, Li, Z, Tang, R, Jia, H, Liu, B., 2011. Novel (E)-5-styryl-2,2 -bithiophene
derivatives as ligands for β-amyloid plaques. Eur. J Med Chem. 46 (7), 2908–2916.
F.A.S. Politi conducted the experiments, analyzed the results and
wrote the manuscript. R.R. Fantatto assisted in the in vitro/in vivo bio-
logical tests. R.V. Bueno assisted in the in silico molecular assays. L. Zeoly
performed the organic syntheses. J.O. Eloy assisted the nanoformulation
preparation. M. Furlan, A.C.S. Chagas, F.A.S. Coelho, R.V.C. Guido and
M. Chorilli conceived the research and reviewed the final manuscript.
De La Ossa, DHP, Ligresti, A, Gil-Alegre, ME, Aberturas, MR, Molpeceres, J, Di Marzo, V,
Torres-Su ´a rez, AI., 2012. Poly-
ε-caprolactone microspheres as a drug delivery system
for cannabinoid administration: Development, characterization and in vitro
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Desany, B, Zhang, Z., 2004. Bioinformatics and cancer target discovery. Drug Disc Today
9 (18), 795–802.
Dunn, SE, Coombes, AGA, Garnett, MC, Davis, SS, Davies, MC, Illum, L., 1997. In vitro
cell interaction and in vivo biodistribution of poly (lactide-co-glycolide) nanospheres
surface modified by poloxamer and poloxamine copolymers. J Control Release 44
(1), 65–76.
Declaration of Competing Interest
Faizi, S, Naz, A., 2002. Jafrine, a novel and labile β-carboline alkaloid from the flowers of
Tagetes patula. Tetrahedron 58 (31), 6185–6197.
The authors declare that there is no conflict of interest.
Fessi, H, Puisieux, F, Devissaguet, JP, Ammoury, N, Benita, S, 1989. Nanocapsule
formation by interfacial polymer deposition following solvent displacement. Int J
Pharm 55 (1), R1–R4.
Acknowledgments
Friesner, RA, Banks, JL, Murphy, RB., et al., 2004. Glide: A new approach for rapid,
accurate docking and scoring. 1. Method and assessment of docking accuracy. J Med
Chem 47 (7), 1739–1749.
We thank S a˜ o Paulo Research Foundation (FAPESP, grant 2013/
Friesner, RA, Murphy, RB, Repasky, MP, Frye, LL, Greenwood, JR, Halgren, TA,
Sanschagrin, PC, Mainz, DT., 2006. Extra precision glide: Docking and scoring
incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med
Chem 49 (21), 6177–6196.
0
3493-8 to FASP, 2013/07600-3/CIBFar to MF and FC, 2011/08042-9
to RVCG, 2013/04737-8 to RVB and 2014/50926-0 (INCT-FAPESP) to
MF, and National Council for Scientific and Technological Development
Frohlich, E., 2012. The role of surface charge in cellular uptake and cytotoxicity of
medical nanoparticles. Int J Nanomed 7, 5577–5591.
(
CNPq), grants 150302/2017-7 to FASP, 304256/2018-8 to RVCG and
65637-0/2014 (INCT-CNPq) for funding the research and fellowships.
4
Gainza, YA, Fantatto, RR, Chaves, FCM, Bizzo, HR, Esteves, SN, Chagas, ACS., 2016. Piper
aduncum against Haemonchus contortus isolates: cross resistance and the research of
natural bioactive compounds. Braz J Vet Parasitol 25 (4), 383–393.
Gainza, YA, Domingues, LF, Perez, OP, Rabelo, MD, L o´ pez, ER, Chagas, ACS., 2015.
Anthelmintic activity in vitro of Citrus sinensis and Melaleuca quinquenervia essential
oil from Cuba on Haemonchus contortus. Ind Crops Prod 76, 647–652.
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