Aedes aegypti is associated with the transmission of numerous human and animal diseases, such as yellow fever, dengue fever, chikungunya, and more recently Zika virus. Emerging insecticide resistance has created a need to develop new mosquitocidal agents for effective control operations. A series of benzothiazole-piperidine derivatives (1-24) were investigated for their larvicidal and adulticidal effects on Ae. aegypti It was observed that compounds 2, 4, 6, 7, 8, 11 and 13 showed notable larvicidal activity. Furthermore, compounds 6 and 10 showed promising adulticidal activity. Based on the mosquitocidal properties of these compounds, docking studies were also carried out in the active site of the AeSCP2 enzyme to explore any insights into further in vitro enzyme studies. Docking results indicated that all these active compounds showed reasonable interactions with critical residues in the active site of this enzyme. This outcome suggested that these compounds might show their larvicidal and adulticidal effects via the inhibition of AeSCP2. According to in vitro and in silico studies, compounds 2, 4, 6, 7, 8, 10, 11 and 13 stand out as candidates for further studies.
Mosquitoes are one of the most dangerous insect vectors and infectious disease carriers in developing countries . Among mosquito species, Aedes aegypti L. transmits yellow fever, dengue fever, chikungunya, and more recently, Zika virus . Dengue is an endemic viral disease found mainly in the tropical and subtropical regions across the globe [3,4]. Dengue is characterized by fever, headache, muscle, and joint pain together with nausea and vomiting [4, 5, 6]. Zika virus also causes severe brain defects and threatens the lives and health of adults and newborns from infected mothers .
The battle against mosquitoes has become a crucial environmental, economic and social health issue. Generally, chemical insecticides are considered as the first option for reducing vector-borne disease but evolving resistance caused by cytochrome P450 monooxygenases (P450s), which are capable of metabolizing many insecticides, as well as decreases in target site sensitivity can limit the success of insecticide treatment [8, 9, 10]. In particular, overexpression of P450s such as the CYP9J32 gene in Ae. aegypti is associated with pyrethroid resistance [11,12].
Sterol carrier protein-2 (SCP-2), a nonspecific intracellular lipid carrier, is expressed throughout the animal kingdom including insects. Moreover, single SCP-2 domain genes have also been proven to be expanded in mosquitoes . Cholesterol is crucial for insects in order to grow, develop and reproduce, but they are not capable of synthesizing cholesterol de novo [14,15]. The mosquito SCP-2, Ae. aegypti SCP-2 (AeSCP2), has been reported to be involved in cholesterol and fatty acid uptake in the midgut in both larval and adult mosquitoes . A small number of studies have focused on developing new insecticides that prevent cholesterol biosynthesis targeting AeSCP2 [17, 18, 19]. Fifty seven compounds were identified in silico as possible inhibitors of the cholesterol-binding capacity of SCP-2 from the library of 16000 compounds . Therefore, targeting this cholesterol transport pathway associated with AeSCP2 could be an alternative target for the development of specific mosquitocidal agents.
Benzothiazole (BT) is a privileged bicyclic ring system present in a wide variety of synthetic and natural products. BT and its derivatives play a distinctive role in medicinal chemistry due to their diverse biological activities such as antiprotozoal, antimicrobial, anticancer, antischizophrenia, antihypertensive, anti-inflammatory, and antiviral activities [21, 22, 23, 24, 25]. Venugopala et al. 2013 , also screened benzothiazole analogs for their mosquitocidal and repellent properties against Anopheles arabiensis by mosquito feeding-probing assay, cone bio-assay and standard World Health Organization (WHO) larvicidal assay. Similarly, piperidine is a strong base mainly found in several natural alkaloid skeletons . Diversely substituted piperidines are the leading heterocycles in the structure of several important pharmaceuticals such as bupivacaine, troxipide, tofacitinib, fexofenadine, astemizole, fentanyl, haloperidol, loperamide, trimeperidine, etc. .
On the basis of these findings, benzothiazole-piperidine derivatives, which had been reported previously for their antimicrobial effects on pathogenic bacteria and Candida species by our research group , were evaluated for their insecticidal activities against Ae. aegypti. In addition, molecular docking studies were performed for the most effective compounds within the active site of AeSCP2 (PDB code: 1PZ4) to provide a mechanistic approach for further studies .
2-Chloro-N-(benzothiazol-2-yl)acetamide derivatives were synthesized via the reaction of 2-aminobenzothiazoles with chloroacetyl chloride in the presence of triethylamine. N-(Benzothiazol-2-yl)-2-(piperidin-1-yl)acetamide derivatives (1-24) (Table 1) were obtained by the nucleophilic substitution reaction of 2-chloro-N-(benzothiazol-2-yl) acetamides with piperidine derivatives in the presence of potassium carbonate. The synthetic protocol and spectral data of the compounds were reported previously by our research group .
2.2 Biological assays
The Orlando 1952 strain (ORL1952) of Ae. aegypti is a laboratory susceptible colony that has been without wildtype supplementation for over seventy years. The strain was originally collected near Orlando Florida, USA and has been maintained by the USDA ARS Center for Medical, Agricultural and Veterinary Entomology (CMAVE) in Gainesville, Florida (previously the Insects Affecting Man Laboratory). Insecticide susceptibility of this strain has been characterized for a large number of common pesticides [31,32]. Colony maintenance and organism rearing for bioassays were described previously .
2.2.2 Larvicidal activity
The larval bioassay was described in detail previously . Briefly, ORL1952 eggs were hatched overnight in approximately 100 mL of deionized water. Five first instar larvae were transferred into the wells of 96-well flat-bottom tissue culture plate in 188 μL of deionized water. Ten microliters of a 2% solution of finely ground alfalfa powder were added to each well.
Compounds 1-24 were dissolved in DMSO to produce a 100 mg/μL stock solution. Dilutions of each stock solution were created by adding 2, 1, 0.5, or 0.2 microliters to the wells containing larva and food media for the initial screening bioassay. Negative control wells were prepared with 2 microliters of DMSO and positive control wells contained permethrin. Bioassay plates were covered and maintained on the benchtop at 22-23°C. Mortality was scored at 24 hours and the assay was repeated on three separate days. Additional dilutions (to 0.01 mg/μL; 0.005 mg/μL for compound 11) of active compounds (>80% mortality at 0.1 mg/μL) were tested to determine the full dose response curve and allow calculation of LC50 values.
2.2.3 Adulticidal activity
Three to seven-day post-emergence female Ae. aegypti were sorted into groups of 10 in TK35 plastic cups after one hour of chilling at 4°C. Compound stock solutions in DMSO (100 μg/μL) were diluted in acetone to produce a 10 mg/μL solution. Five hundred nanoliters of the compound solution were applied to the thorax of each mosquito using a repeating syringe with a blunt tip needle (Hamilton PB600 with 7100 syringe). Cups containing treated mosquitoes were capped with tulle mesh and mosquitoes were allowed to recover with access to 10% sucrose saturated cotton balls. Mortality was recorded at 24 hours and the assay was repeated at least three times. Acetone was used as a negative control, whereas permethrin (mixture of 46.1% cis and 53.2% trans isomers (Chemservice, West Chester, PA, USA)) was used as a positive control. Additional dilutions (from 5 ug/mosq to 0.5 ug/mosq) and assays of active compounds were performed to calculate LD50 values. Specific details of the adult topical bioassay were published previously .
2.2.4 LD50 and LC50 calculation
Median lethal doses were calculated using SigmaPlot (v13). Raw mortality counts from each dose of each replicate were converted to percentage mortality and plotted. Curve fitting of dose and mortality data was performed using a four parameter logistic model with constrained minimum and maximum values of 0 and 100, respectively.
2.3 Molecular docking studies
Molecular docking studies were carried out to understand the relationship between protein structures and substrates, which can provide reasonable explanations for substrate specificities and differences in the active site of the target structure. As previously mentioned, inhibition of AeSCP2 can reduce the uptake of cholesterol and lead to death in both larval and adult mosquitoes. For this purpose, compounds 2, 4, 6, 7, 8, 10, 11 and 13 were docked to the active site of AeSCP2 due to their in vitro mosquitocidal potencies. Ligands were sketched and cleaned in Maestro molecular modeling workspace followed by energy minimization in ligand preparation program of Schrödinger’s Maestro molecular modeling package (Schrödinger Release 2016-2: Schrödinger, LLC, New York, NY, USA) using Optimized Potential Liquid Simulations (OPLS_2005) force field. The X-ray crystallographic structure of the AeSCP2 complex with palmitic acid (PDB code: 1PZ4)  was retrieved from the Protein Data Bank (PDB) server and optimized for docking analysis in protein preparation module of Schrödinger software. In molecular docking simulations, Glide/XP docking protocols were applied for the prediction of topologies of active compounds in the active site of AeSCP2.
Ethical approval: The conducted research is not related to either human or animal use.
3 Results and Discussion
Compounds 1-24 were investigated for their larvicidal and adulticidal activities against Ae. aegypti Initial screening of the first instar larvae was completed at four concentrations (1.0, 0.5, 0.25, 0.1 mg/μL) with seven compounds producing more than 80% mortality at the 0.1 mg/μL dose (Table 2). Compounds 6 and 10 produced more than 80% mortality in initial screening of adult Ae. aegypti females (Table 2). LC50 or LD50 values were subsequently determined for compounds that produced
|#||Larvicidal activity against 1st instar Ae. aegypti*||Adulticidal activity against adult female Ae. aegypti**|
|1 mg/μL||0.5 mg/μL||0.25 mg/μL||0.1 mg/μL||LC50 ± SE (mg/μL)||IC50 (μM)||R2||5 mg/ mosquito||LD50 ± SE (mg/mosq.)||LD50 (μM)||R2|
|1||93 ± 12||87 ± 12||100||73 ± 31||-||-||64 ± 6||-||-|
|2||100||100||100||100||0.096 ± 0.005||332.2||0.984||47 ± 6||-||-|
|3||73 ± 12||40 ± 20||0||0||-||-||47 ± 29||-||-|
|4||100||100||100||87 ± 23||0.065 ± 0.011||214.5||0.794||60 ± 27||-||-|
|5||100||93 ± 12||87 ± 23||60 ± 53||-||-||77 ± 21||-||-|
|6||100||100||100||100||0.053 ± 0.011||163.8||0.643||83 ± 12||2.084 ± 0.338||6442.0||0.926|
|7||100||100||93 ± 12||93 ± 12||0.128 ± 0.019||379.2||0.803||47 ± 29||-||-|
|8||100||100||100||87 ± 23||0.123 ± 0.015||364.4||0.981||63 ± 12||-||-|
|9||47 ± 42||47 ± 42||33 ± 31||33 ± 58||-||-||70||-||-|
|10||100||87 ± 12||80 ± 20||67 ± 12||-||-||83 ± 6||2.962 ± 0.670||9775.6||0.533|
|11||100||100||100||100||0.034 ± 0.003||107.3||0.647||40 ± 17||-||-|
|12||100||87 ± 23||73 ± 46||67 ± 58||-||-||57 ± 15||-||-|
|13||100||100||100||100||0.098 ± 0.001||307.2||0.958||67 ± 25||-||-|
|14||100||100||100||73 ± 23||-||-||50 ± 36||-||-|
|15||55 ± 22||40 ± 0||25 ± 7||15 ± 7||-||-||73 ± 12||-||-|
|16||100||100||73 ± 46||67 ± 58||-||-||53 ± 12||-||-|
|17||100||73 ± 31||47 ± 50||67 ± 58||-||-||63 ± 15||-||-|
|18||100||93 ± 12||87 ± 23||47 ± 31||-||-||37 ± 6||-||-|
|19||100||93 ± 12||80 ± 35||67 ± 31||-||-||67 ± 12||-||-|
|20||65 ± 21||45 ± 7||30 ± 14||15 ± 7||-||-||50 ± 10||-||-|
|21||93 ± 12||53 ± 50||33 ± 58||33 ± 58||-||-||50 ± 30||-||-|
|22||93 ± 12||53 ± 50||33 ± 58||33 ± 58||-||-||63 ± 6||-||-|
|23||33 ± 31||20 ± 20||27 ± 46||7 ± 12||-||-||73 ± 12||-||-|
|24||100||100||93 ± 12||67 ± 31||-||-||73 ± 12||-||-|
* Positive control permethrin at 3.0 pg/μL resulted in 26.7 ± 23.1 % mortality. Negative control solvent control (DMSO) had 0% mortality.
** The average mortality in the two permethrin positive controls of 0.1935 and 0.4772 ng/mosquito was 63.3 ± 15.3 and 100, respectively. Acetone controls had 0% mortality.
>80% mortality at the discriminating dose in either or both bioassays by further subdilutions and additional assays (Table 2). Compounds 11, 6, 4, 2, 13, 8, and 7 showed the highest larvicidal activity with LC50 values of 0.034 (107.3 μM), 0.053 (163.8 μM), 0.065 (214.5 μM), 0.096 (332.2 μM), 0.098 (307.2 μM), 0.123 (364.4 μM), and 0.128 (379.2 μM) μg/μL, respectively. Compounds 6 and 10 exhibited the highest adulticidal activity with LD50 values of 2.084 (6442.0 μM) and 2.962 (9775.6 μM) μg/mosquito, respectively. Compounds 1, 3, 5, 9, 12, 14-24 did not show strong larvicidal or adulticidal activity.
Generally, 4-methyl substitution on the piperidine ring and 6-methoxy substitution on the benzothiazole ring, apart from compound 13, caused the loss of larvicidal activity. On the other hand, 6-chloro substituted benzothiazoles (6, 7 and 8) displayed reasonable larvicidal activity; only compound 5 did not possess any antimosquito properties related to 4-methyl substitution on the piperidine ring. This outcome indicated the importance of the chloro substitution at the sixth position of the benzothiazole scaffold. Among nonsubstituted benzothiazoles (1-4), only 3-methyl substituted piperidine-based compound 2 and 3,5-dimethyl substituted piperidine-based compound 4 showed significant larvicidal activity, indicating that the methyl substitution at the third position of the piperidine ring enhanced the larvicidal activity. The presence of 3-methyl moiety on piperidine ring also influenced adulticidal activity positively as observed in compounds 6 and 10. In general, the loss of larvicidal activity was detected with 6-ethoxy and 6-nitro substitutions on the benzothiazole ring as observed in compounds 17-24.
According to docking results, compounds 2, 4, 6, 7, 8, 10, 11, and 13 showed high affinity and substrate-specific interactions in the active site of AeSCP2 [30,34] (Figure: 1). Benzothiazoles of compounds 2, 6, 7, 10, 11, and 13 presented π-π interactions with Arg15 and Phe105 residues of the binding site of AeSCP2. The methoxy substitution on the benzothiazole ring of compound 13 could account for all interactions of this compound. Piperidines of compounds 4, 6, 7, 8, 10 and 11 displayed cation-π interaction with the Arg15 residue and only 4-methyl substitution on the piperidine ring did not have a positive influence. All interactions of compounds 4 and 8 were related to 3,5-dimethyl substitution on the piperidine ring. Moreover, the acetamido moieties of compounds 2 and 10 served as H-bond donors for the in pocket residue Leu102. The docking score, glide gscore and glide emodel results of compounds 2, 4, 6, 7, 8, 10, 11, and 13 are also given in Table 3. The emodel score is appropriate for the comparison of different conformations of the same ligand, but the docking score is generally used for the comparison of different ligands . The docking scores of the compounds were determined to range between -8.19 and -8.95 kcal/mol as similar to each other.
|Compound||Docking score||Glide gscore||Glide emodel|
In the present work, compounds carrying benzothiazole and piperidine rings were investigated for first instar larvicidal activity and adulticidal activity against Ae. aegypti. The most potent larvicidal and adulticidal compounds in this series were also analyzed for molecular docking interactions in the active site of the AeSCP2 to provide mechanistic insight for further in vitro enzyme studies. According to mosquitocidal assays, compounds 2, 4, 6, 7, 8, 11 and 13 were found to be the most promising larvicidal agents, whereas compounds 6 and 10 were identified as the most promising adulticidal agents. Based on the results, 3-methyl substitution on the piperidine ring enhanced the larvicidal and adulticidal activity. Besides, 6-chloro substitution on the benzothiazole ring also increased the larvicidal activity. The docking results of compounds 2, 4, 6, 7, 8, 10, 11 and 13 suggested that π-π interactions and hydrogen bonds were responsible for the observed affinity in the active site of the AeSCP2. This outcome supported the hypothesis that the larvicidal and adulticidal activities of these compounds could result from the inhibition of AeSCP2.
This study was partly funded by the Deployed War-Fighter Protection Research Program via grants from the U.S. Department of Defense through the Armed Forces Pest Management Board (to JJB and to JRB). We thank Miss Jessica Louton (USDA-ARS, CMAVE, Gainesville, FL) for mosquito bioassays. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture or the U.S. Department of Defense. USDA is an equal opportunity provider and employer.
Conflict of interest: The authors declare that they have no conflict of interest.
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