Transgenic Drosophila melanogaster insects and genetics
Drosophila melanogaster flies were reared on standard agar-cornmeal medium at 25 °C under a 12 h:12 h light-dark cycle. The noboKO strain used has been previously described [23]. phm-GAL4#22 (Research Resource Identifier (RRID):BDSC_80577) [56, 57] was kindly gifted by Michael B. O’Connor (University of Minnesota, USA).
The GAL4/UAS system [58] was used to overexpress the AeNobo gene in D. melanogaster. pUAST-attB plasmid [59], carrying the AeNobo coding sequence, was built using the custom gene synthesis service of VectorBuilder, Inc. Transformants were generated using the φC31 integrase system in the P{CaryP}attP40 strain (RRID:BDSC_79604) [60]. The w+ transformants of pUAST-attB were established using standard protocols. Viability of noboKO animals, expressing the nobo transgene driven by phm-GAL4#22, was examined as described previously [23].
Flavonoid chemicals
The following flavonoids were used in this study: biochanin A (>98%, Tokyo Chemical Industry, B4098), (+)-catechin hydrate (98%, Tokyo Chemical Industry, C0705), chrysin (98%, Alfa Aesar, L14178), cyanidin chloride (98%, Fujifilm Wako Chemicals, 030-21961), daidzein (DAI; ≥98%, Nagara Science, NH010102), desmethylglycitein (DMG; >95%, Tokyo Chemical Industry, T3473), S-equal (≥97%, Merck, SML2147), fisetin (>96%, Tokyo Chemical Industry, T0121), genistein (>98%, Tokyo Chemical Industry, G0272), 2′-hydroxyflavanone (>98%, Tokyo Chemical Industry, H1024), kaempferol (≥98%, Cayman Chemical Company, 11852), luteolin (95%, Fujifilm Wako Chemicals, 127-06241), myricetin (>97%, Tokyo Chemical Industry, M2131), naringenin (>98%, Alomone Labs, N-110), petunidin (≥98%, Cayman Chemical Company, 19755), quercetin (96.5%, Fujifilm Wako Chemicals, 512-58344), tamarixetin (>99%, Extrasynthese, 1140S), 5,3′,4′-trihydroxyflavone (>85%, Toronto Research Chemicals, T896685), and 7,3′,4′-trihydroxyflavone (>85%, Toronto Research Chemicals, T896780).
Estrogenic compounds
The following estrogenic compounds were used in this study: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (≥98%, Sigma-Aldrich, 22590), 1-benzyl 2-butyl benzene-1,2-dicarboxylate (98%, eNovation, D584181), 16-benzylidene estrone (95%, OTAVA, 7569822), Biochanin A (>90%, InterBioScreen, BB_NC-02653), Biosphenol A (4,4′-propane-2,2-diyldiphenol) (90%, Vitas-M, STK801675), bis(2,4-dihydroxyphenyl)methanone (>90%, ChemBridge, 5222210), butyl 4-hydroxybenzoate (>90%, ChemDiv(LB), 0099-0145), 1,1-Dichloro-2,2-bis(4-chlorophenyl)ethene (90%, Sigma-Aldrich, 123897), diethylstilbestrol (4-[(E)-4-(4-hydroxyphenyl)hex-3-en-3-yl]phenol) (>98%, Tokyo Chemical Industry, D0526), Ferutinine (>90%, InterBioScreen, STOCK1N-32042), (S)-5-(4-hydroxy-3,5-dimethylphenyl)-1-methyl-2,3-dihydro-1H-inden-1-ol (90%, Sigma-Aldrich, SML1876), 4-[(1S,5R)-5-(hydroxymethyl)-8-methyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]phenol (>90%, InterBioScreen, STOCK1N-10587), 4-[(1R,5R)-5-(hydroxymethyl)-6,8,9-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]phenol (>90%, InterBioScreen, STOCK1N-13438), 4-[(1R,5R)-4,4,8-trimethyl-3-oxabicyclo[3.3.1]non-7-en-2-yl]phenol (>90%, InterBioScreen, STOCK1N-00708), propyl 4-hydroxybenzoate (>90%, ChemDiv(LB), 0099-0143), Resveratrol (>90%, InterBioScreen, BB_NC-02570), 4-[2,2,2-trichloro-1-(4-hydroxyphenyl)ethyl]phenol (90%, Vitas-M, STK996193), 4-(2,4,4-trimethylpentan-2-yl)phenol (>90%, Chemspace, PB425466960), α-zearalanol (>90%, InterBioScreen, STOCK1N-99337), and Zearalenone (>90%, InterBioScreen, STOCK1N-03962).
Plasmid construction of Escherichia coli protein expression system
Aedes aegypti (SMK strain, 4th instar larvae, eight animals) cDNA was obtained by reverse transcription using ReverTra Ace qPCR RT Master Mix (Toyobo). AeNobo coding region was amplified from Ae. aegypti cDNA using PCR. The forward primer 5′-ATGTCCAAACCGGTGCTGTATTAC-3′ and the reverse primer 5′-CTATTTTTTCATTACAGCATGAAGTCTC-3′ were used for touchdown PCR. Touchdown PCR was conducted as follows: first, denaturation was performed for 10 s at 98 °C, followed by annealing for 30 s at 68 °C and extension for 1 min at 68 °C for 13 cycles. Next, denaturation was performed for 10 s at 98 °C, followed by annealing for 30 s at 55 °C and extension for 1 min at 68 °C for 30 cycles. The PCR product was ligated to pBluescript SK(-) to check and confirm the AeNobo sequence. After sequence verification, AeNobo was amplified from the vector through PCR using KOD-plus Neo polymerase (Toyobo) under the following conditions: pre-denaturation at 94 °C for 2 min, denaturation at 98 °C for 10 s, and extension at 68 °C for 30 s for 40 cycles. The product was extracted and ligated to pCold III (Takara Bio) for expression of the AeNobo-WT protein in Escherichia coli.
pCold III plasmids expressing the AeNobo-E113A and AeNobo-F39L were constructed using KOD-Plus-Mutagenesis Kit (Toyobo). The following primers were used: E113A-F (5′-CGTCGATCGTAATGCGAGGCTTGATC-3′) and E113A-R (5′-CTCTTTGGAACAGAACGGCATTGTTG-3′) for AeNobo-E113A and F39L-F (5′-AGAGAGAGAACATCTTTTGGAAG-3′) and F39L-R (5′-AAGAGGCGAACCAGTTTGAGTTC-3′) for AeNobo-F39L. We conducted inverse PCR using KOD-plus polymerase, pColdIII/AeNobo-WT plasmid, and the primer pair described above under the following conditions: pre-denaturation at 94 °C for 2 min, denaturation at 98 °C for 10 s, and extension at 68 °C for 6 min for 6 cycles. The PCR products were treated with DpnI at 37 °C for 1 h, followed by self-ligation using T4 polynucleotide kinase and Ligation High. After the transformation of DH5α bacteria using the ligation products, we extracted plasmid DNA from the colonies and verified their sequences to confirm whether the appropriate point mutations were introduced.
Protein expression and purification
Recombinant DmNobo protein was produced using an E. coli expression system as previously described [29, 30]. Similarly, recombinant AeNobo protein was produced using an E. coli expression system as follows: pCold III-AeNobo plasmid was transformed into the E. coli BL21 Star (DE3) strain (Thermo Fisher Scientific) for 30 min at 4 °C. The transformant was plated in Luria-Bertani (LB) medium supplemented with 100 μg/mL ampicillin and incubated at 37 °C overnight. Next, a bacterial colony from the plate was inoculated into 200 mL of LB supplemented with 100 μg/mL ampicillin (LB-amp medium) and shaken at 37 °C overnight for preculture. The preculture was transferred to 6 L of LB-amp medium for the main culture and shaken at 37 °C. After the optical density of the culture reached 1.0, Nobo protein expression was induced by incubation with 0.1 mM isopropyl β-D-1-thiogalactopyranoside at 15 °C overnight. Next, the bacterial cells were harvested via centrifugation at 4,000 × g for 15 min. The bacterial pellet was stored at −80 °C. The pellet from the 3-L culture was suspended in a lysis buffer (140 mM NaCl, 20 mM Tris-HCl at pH 8.0, and 1 mM dithiothreitol [DTT]). The cells were disrupted via sonication for 2 min at 70% amplitude with output 7 using the ULTRA5 HOMOGENIZER VP-305 (TAITEC) on ice. The soluble lysate was fractionated using centrifugation at 35,000 × g for 30 min. The supernatant was mixed with 10 mL of Glutathione Sepharose 4B beads (Cytiva) for 1 h at 4 °C for glutathione affinity purification. The beads were then collected and washed in lysis buffer. Proteins bound to the beads were eluted using 50 mL of elution buffer (10 mM GSH, 140 mM NaCl, 20 mM Tris-HCl at pH 8.0, and 1 mM DTT). Next, the eluent was concentrated to 5 mL and fractionated using size exclusion column chromatography with a HiLoad Superdex200 26/60 instrument (Cytiva) equilibrated using a size exclusion buffer (150 mM NaCl, 25 mM Tris-HCl at pH 8.0, and 5 mM DTT) at a flow rate of 1 mL/min. The purity of the fractions was evaluated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining. Peak fractions were collected, and their buffer was replaced with another buffer (150 mM NaCl, 25 mM Tris-HCl at pH 8.0, 5 mM DTT, 10 mM GSH) by ultrafiltration conducted twice with an Amicon Ultra-15 30,000 MWCO instrument (Merck); proteins were then concentrated to 45 mg/mL. Protein concentration was measured by spectrophotometry using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) at an extinction coefficient (ε280) of 0.852 M−1 cm−1. Finally, the protein was stored at −80 °C.
Measurement of specific activity of AeNobo protein in vitro
In vitro GST assays using 3,4-DNADCF were performed as described previously [29]. The stock solutions of AeNobo-WT, AeNobo-E113A, and AeNobo-F39L were 174.6, 227.8, and 291.4 ng/mL, respectively, in solution A (2 mM GSH, 100 mM sodium phosphate buffer at pH 6.5, 0.01% Tween20). Decreasing concentrations of AeNobo-WT, AeNobo-E113A, and AeNobo-F39L, ranging from 174.6 to 3.0 ng/mL, from 227.8 to 4.0 ng/mL, and from 291.4 to 5.0 ng/mL, respectively, were prepared by 2/3-fold serial dilution with solution A. The AeNobo dilution series was mixed with an equal volume of solution B (100 mM sodium phosphate buffer at pH 6.5, with 2mM 3,4-DNADCF in 0.2% dimethyl sulfoxide (DMSO) as a co-solvent) in each well of a 96-well plate to initiate the catalytic reaction of DmNobo. In these wells, the final concentrations of AeNobo-WT, AeNobo-E113A, and AeNobo-F39L ranged from 87.3 to 1.5 ng/mL, from 113.9 to 2.0 ng/mL, and from 145.7 to from 2.5 ng/mL, respectively. The glutathione-conjugated product was excited at 485 nm wavelength, and the fluorescence intensity at 535 nm or 538 nm wavelength (Fmeasured) was measured every 30 s for 20 min using an Infinite 200 PRO instrument (Tecan) or Fluoroskan Ascent™ FL (Thermo Fisher Scientific). The specific activity of AeNobo enzymes was determined as previously described [30].
GST activity inhibition assay
The IC50 value was measured using an in vitro assay system as described previously [29]. A dilution series of compounds, ranging from 2.5 mM to 0.127 μM for DMG and from 2.5 mM to 4.9 μM for other compounds, was prepared by 2-fold serial dilution in DMSO. Five microliters of each diluted compound solution was mixed with 245 μL of solution A (100 mM sodium phosphate buffer at pH 6.5, 0.01% Tween 20, 2 mM GSH, and 50 ng/mL AeNobo-WT, 100 ng/mL AeNobo-E113A, and 200 ng/mL F39L). One hundred microliters of the mixture was dispensed into wells of a 96-well plate. One hundred microliters of solution B (0.2 μM 3,4-DNADCF and 100 mM sodium phosphate buffer at pH 6.5) was added to each well. In summary, the final reaction system comprised 100 mM sodium phosphate buffer (pH 6.5), 1 mM GSH, 0.005% Tween 20, 0.1 μM 3,4-DNACDF, and 25 ng/mL AeNobo, 50 ng/mL AeNobo-E113A, or 100 ng/mL AeNobo-F39L protein. The fluorescence intensity derived from 4-GS-3-NADCF, a product of this reaction, was measured for 3 min. IC50 values were estimated as described previously [30]. The enzymatic assays under each condition were performed at least twice independently.
High-throughput screening of 9600 compounds
To identify inhibitors of AeNobo enzymatic activity, a high-throughput screening was performed as described in a previous study [29]. Briefly, a core library of 9600 compounds obtained from the Drug Discovery Initiative, The University of Tokyo, was utilized for screening. In this screen, the enzymatic activity of AeNobo was detected using 3,4-DNADCF [29]. First, 1 μL of solution A (11.2 ng/mL AeNobo protein, 100 mM sodium phosphate at pH 6.5, 2 mM GSH, and 0.005% Tween 20) was dispensed into each well of a 1536-well plate (1536 Black SV/NB/FI, #784900, Greiner Bio-One) together with 0.01 μL of compounds at a concentration of 2 mM. Then, 1 μL of solution B (4 μM 3,4-DNADCF, 100 mM sodium phosphate at pH 6.5, and 0.005% Tween 20) was added into each well. In summary, the reaction system comprised 5.6 ng/mL AeNobo protein, 2 μM 3,4-DNADCF, 1 mM GSH, 0.005% Tween 20, and 100 mM sodium phosphate at pH 6.5, together with each compound at 10 μM. The plate was incubated for 30 min at 25–27 °C and 2 μL of 10 mM N-ethylmaleimide was added into each well to stop the reaction. For the first screen, 90 compounds that inhibited the enzymatic activity of AeNobo by more than 50% were selected. For the second screen, we assayed the IC50 values of the compounds against the enzymatic activity of AeNobo. Compounds with IC50 values lower than 10 μM were defined as hit compounds.
Crystallization
A 100 mM GSH stock solution was prepared in a buffer composed of 150 mM NaCl, 25 mM Tris-HCl at pH 8.0, and 5 mM DTT. The GSH stock solution was diluted to 30 mM GSH by adding an AeNobo protein solution for co-crystallization. The AeNobo protein solution was centrifuged at 13,500 × g for 30 min to remove protein aggregates.
An initial crystallization assay was performed using a Protein Crystallization System (PXS) [61] following the sitting drop vapor diffusion crystallization method with 0.2 μL of each protein solution and one of the reservoir solutions from the following kits: Crystal Screen 1 & 2 (Hampton Research, Aliso Viejo, CA, USA), Index (Hampton Research), PEGIon (Hampton Research), PEGIon2 (Hampton Research), Wizard I & II (Molecular Dimensions, Suffolk, UK), PEGs II Suite (Qiagen), Protein Complex Suite (Qiagen), Stura FootPrint Screen (Molecular Dimensions), and MembFac (Hampton Research) at 20 °C. Under these conditions, crystals formed only when the reservoir solution was composed of 30% (w/v) PEG 4000, 0.1 M Tris-HCl at pH 8.5, and 0.2 M magnesium chloride. The conditions were optimized using the hanging drop vapor diffusion method, through which crystals formed in drops of 1 μL, each containing 45 mg/mL AeNobo protein and a reservoir solution (32.5% (w/v) PEG 4000, 0.1 M Tris-HCl, pH 7.5, 0.5 M calcium chloride) at 20 °C. To obtain a structure complexed with flavonoids, AeNobo crystals were soaked in 30 mM luteolin or DMG suspensions in the reservoir solution for 1 day at 20 °C. As luteolin did not completely dissolve in the reservoir solution at 30 mM, AeNobo crystals were soaked in a reservoir solution saturated with luteolin for 1 day at 20 °C.
X-ray crystallography
Crystals were soaked in a cryoprotectant solution (30% (w/v) Polyvinylpyrrolidone K 15 Average Molecular Wt. 10000 (Tokyo Chemical Industry, P0471)/reservoir solution), picked with cryo-loops (MiTeGen), flash frozen in liquid nitrogen, and packed in Uni-pucks (Molecular Dimensions). X-ray diffraction experiments for crystals of AeNobo-GSH, AeNobo-GSH-daidzein, AeNobo-GSH-luteolin, and AeNobo-GSH-DMG complexes were performed at beamlines BL-17A, BL-5A, NE-3A, and BL-5A, respectively, at the Photon Factory, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan. The collected datasets were processed and scaled using XDS (RRID:SCR_015652) [62] and AIMLESS (RRID:SCR_015747) [63], respectively. Space groups were determined using POINTLESS (RRID:SCR_014218) [64]. Phases for AeNobo-GSH were calculated using the molecular replacement method with the DmNobo structure (PDB ID: 6KEM) [37] as a template, and those for AeNobo-GSH-daidzein, AeNobo-GSH-luteolin, and AeNobo-GSH-DMG were calculated using the AeNobo-GSH structure. Model building and crystallographic refinement were performed by COOT (RRID:SCR_014222) and PHENIX.REFINE (RRID:SCR_016736) [65, 66]. The crystallographic statistics are summarized in Additional file 2: Table S3.
MD simulation
The structures of AeNobo-GSH-daidzein and AeNobo-GSH-DMG were processed to assign bond orders and hydrogenation. The ionization states of each compound and GSH at pH 7.0 ± 2.0 were predicted using Epik [67], and H-bond optimization was conducted using PROPKA [68]. Energy minimization was performed in Maestro (Schrödinger) using the OPLS3e force field [69]. Each E113A mutation model for MD simulation was constructed in Maestro and treated using the same protocol. MD simulations were prepared using the Molecular Dynamics System Setup Module of Maestro. All structures were subjected to energy minimization and placed in an orthorhombic box with a buffer distance of 10 Å to create a hydration model, and the SPC water model [70] was used for the hydration model. NaCl (0.15 M) served as the counter ion to neutralize the system. The MD simulations were performed using the Desmond software, version 2.3 (Schrödinger) (RRID:SCR_014575). The cutoff radii for van der Waals and the time step, initial temperature, and pressure of the system were set to 9 Å, 2.0 femtoseconds, 300 K, and 1.01325 bar, respectively. The sampling interval during the simulation was set to 100 ps. Finally, we performed MD simulations using the NPT ensemble for 1 μs. All trajectories from MD simulations were aligned to the initial structure with protein Cα, and ligand RMSD values were calculated based on ligand heavy atoms.
Mosquito rearing
The Ae. aegypti strain used in this study, which originated from the Liverpool strain, was a gift from Ryuichiro Maeda (Obihiro University of Agriculture and Veterinary Medicine). Five hundred pupae were harvested in a plastic cup and placed within a nylon mesh cage (bottom 27 cm × 27 cm, top 25 cm × 25 cm, height 27 cm). A 50-mL glass flask, inserted with a filter paper (#1001-125, Whatman), containing 10% sucrose solution was placed in the nylon mesh cage. The cage rearing the emerged adults was kept in an incubator (MIR-254-PJ, Panasonic Co.) set at 27 °C with humidity over 90% in a standard 12 h:12 h light-dark cycle. The sucrose solution was changed every 3–4 days. Adult females (at 7–14 days after eclosion) were blood fed and allowed to lay eggs on a wet filter paper, 3 to 4 days after engorgement. Eggs laid on the filter paper were washed once with RO water and kept in a plastic container with wet paper for further egg maturation. After a week, the lid of the container was left slightly open to slowly dry the eggs for storage.
Aedes aegypti larvicidal assay
The dried eggs on filter papers were soaked in distilled water. Three hours after soaking, the first instar larvae were transferred to 30 mL of fresh distilled water in a 50-mL plastic cup with a lid containing air holes. In the rearing water, 2 mg of the powdered goldfish food (Hikari Medium Grain, Kyorin Co., Ltd.) was added to each cup as food for Ae. aegypti larvae. In each cup, we poured the 30 mL rearing water containing 1 ppm, 10 ppm, or 100 ppm of the compounds with 0.1% ethanol at the final concentration, followed by placing 20 larvae. Twenty-four hours after the addition of flavonoids, the number of living and dead larvae was recorded. Larvacidal assays under each condition were performed 5 times independently. The larval instars 24 h after the addition of DMG were identified by measuring the transverse diameter of the Ae. aegypti larval head [39]. Photographs of whole bodies of larvae were taken and the head diameter was then measured using ImageJ software [71].
Reverse transcription-quantitative PCR (RT-qPCR)
Preparation of control and DMG-treated larvae was conducted as described above in the previous section “Aedes aegypti larvicidal assay.” Twenty-four hours after the addition of flavonoids, 20-30 Ae. aegypti larvae for each sample were homogenized in RNAiso Plus (Takara Bio Inc.) to extract total RNA. RNA was reverse transcribed to synthesize cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). cDNA samples were used as templates for qPCR using THUNDERBIRD SYBR qPCR Mix (Toyobo) on a Thermal Cycler Dice Real Time System (Takara Bio Inc.). The mRNA level of E74B was normalized to that of an endogenous control ribosomal protein 49 gene (rp49), and the relative fold change was calculated. The normalized E74B expression level was compared using the ΔΔCt method. The primers for E74B were AeaegE74B-Fwd (5′-GCCTTGGAATTCCACTCACAAA-3′) and AeaegE74B-Rev (5′-GGTCTGGTGAACGGACTACACC-3′). The primers for rp49 were Aeaeg-rp49-Fwd (5′-TCGGCAGTCTTGCCAACCCTGA-3′) and Aeaeg-rp49-Rev (5′-AGCTTATCATACCGACGTTCCGAA-3′).
Drosophila melanogaster survival assay
A 0.1% (w/v) DMG stock solution was prepared in 100% DMSO. Ten microliters of the DMG stock solution or 10 μL of DMSO alone was mixed with 10 g of standard cornmeal medium and 1 mL of autoclaved water. The volume of the mixed food was approximately 10 mL, and therefore, the food contained 0.1% DMSO with or without 10 ppm DMG. Two grams of the food were dispensed into each 12-mL plastic vial (Sarstedt). D. melanogaster wild-type Canton-S were given the opportunity to lay eggs on a grape juice agar with yeast paste for 12 h. After egg collection, the embryos were reared at 25 °C for 24 h, and then 20 1st instar larvae were transferred to each vial and reared at 25°C. The numbers of pupae were counted daily, and the timing of pupation was recorded.