An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria

Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects.


Tl + flux assay development
Tl + flux assays were performed essentially as described previously (1). Briefly, stably transfected T-Rex-HEK-293-AnKir1 cells were cultured overnight in 384-well plates in media containing DMEM, 10% dialyzed FBS and 1μg/mL tetracycline to induce channel expression Hamamatsu, Tokyo, Japan) where 20 μL/well of test compounds in assay buffer (as prepared below) were added and allowed to incubate with the cells for 20 min. After the incubation period, a baseline recording was collected at 1 Hz for 10 s (excitation 470 ± 20 nm, emission 540 ± 30 nm) followed by a thallium stimulus buffer addition (10 μL/well) and data collection for an additional 4 min. The Tl + stimulus buffer contains in (mM) 125 NaHCO 3 (Fig. S1B). The robustness and reproducibility of the assay was determined by comparing Tl + flux through tetracycline-induced and tetracyclineuninduced cells (Fig. S1C). The Z' value was calculated as described previously (1), using the following formula: 2 Z' = 1-(3SD p + 3SD n )/|mean p + mean n | where SD is standard deviation, p and n are control and uninduced flux values respectively.
To compare the effect of DMSO on AnKir1-mediated Tl + flux, a one-way ANOVA was performed with a Tukey's multiple comparison test. Prism software (GraphPad Software) was used to generate CRC from Tl + flux. . Half-inhibition concentration (IC 50 ) values were calculated from fits using a four parameter logistic equation.

High-throughput screening
The test compounds were transferred to daughter polypropylene 384-well plates (Greiner Bio-One, Monroe, NC) using an Echo555 liquid handler (Labcyte, Sunnyvale, CA), and then diluted into assay buffer to generate a 2X stock in 0.6% DMSO (0.3% final). For Tl + flux assays on Kir6.2/SUR1 expressing cells, test compounds were diluted in assay buffer containing diazoxide (250 μM final) to induce channel activation. Concentration-response curves (CRCs) were generated by screening compounds at 3-fold dilution series (1 nM -30 μM).
Tl + flux data were analyzed as previously described (2, 3) using a combination of Excel (Microsoft Corp, Redmond, WA) with XLfit add-in (IDBS, Guildford, Surrey, UK), OriginPro (OriginLab, Northampton, MA), and GraphPad Prism (GraphPad Software, San Diego, CA, USA) software. Raw data were opened in Excel and each data point in a given trace was divided by the first data point from that trace (static ratio) followed by subtraction of data points from control traces generated in presence of vehicle controls. The slope of the fluorescence increase beginning 5 s after Tl + addition and ending 15 s after Tl + addition was calculated.

Patch clamp electrophysiology
Patch electrodes were pulled from silanized 1.5 mm outer diameter borosilicate microhematocrit tubes using a P-1000 Flaming/Brown micropipette puller (Sutter Instrument, Novato CA, USA). Electrode resistance ranged from 2-4 MΩ. Whole-cell currents were recorded under voltage-clamp conditions using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Electrical connections to the amplifier were made using Ag/AgCl wires and 3 M KCl/agar bridges. Electrophysiological data were collected at 5 kHz and filtered at 1 kHz.
Data acquisition and analysis were performed using pClamp 9.2 software (Axon Instruments).
Electrophysiology experiments were performed exactly as described previously (1, 4). The compositions of the solutions used for electrophysiology experiments are shown in Table S4. To measure the inhibition of Kir channel activity by VU041, we focused on the maximal inward currents elicited, which occur at the clamp voltage of -140 mV during the voltagestepping protocol. The background current of an oocyte in solution II (low K + ) was subtracted from that in 1) solution III (elevated K + ) to calculate the inward current before exposure to VU041 (I A ), and 2) solution III with VU041 to calculate the inward current after exposure to a small molecule (I B ). The percent inhibition of I A by VU041 was calculated by subtracting I B from I A and then dividing by I A .

Chemical synthesis
All NMR spectra were recorded on a 400 MHz FT-NMR DRX-400 FT-NMR spectrometer or 500 MHz Bruker DRX-500 FT-NMR spectrometer. 1 H chemical shifts are reported in δ values in ppm downfield with the deuterated solvent as the internal standard. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), integration, coupling constant (Hz). High resolution mass spectra were recorded on a Waters Q-TOF API-US plus Acquity system with electrospray ionization. Reversed-phase LCMS analysis was performed using an Agilent 1200 system comprising a binary pump with degasser, high-performance autosampler, thermostatted column compartment, diode-array detector (DAD) and a C18 column. Flow from the column was split to a 6130 SQ mass spectrometer and Polymer Labs ELSD. The MS detector was configured with an electrospray ionization source. Data acquisition was performed with Agilent Chemstation and Analytical Studio Reviewer software. Samples were separated on a ThermoFisher Accucore C18 column (2.6 um, 2.1 x 30 mm) at 1.5 mL/min, with column and solvent temperatures maintained at 45 C. The gradient conditions were 7% to 95% acetonitrile in water (0.1% TFA) over 1.1 minutes. Low-resolution mass spectra were acquired by scanning from 135 to 700 AMU in 0.25 seconds with a step size of 0.1 AMU and peak width of 0.03 minutes. Drying gas flow was 11 liters per minute at a temperature of 350 C and a nebulizer pressure of 40 psi. The capillary needle voltage was 3000 V, and the fragmentor voltage was 100V. Preparative purification was performed on a custom HP1100 purification system (reference 16) with collection triggered by mass detection. Solvents for extraction, washing and chromatography were HPLC grade. All reagents were purchased from Aldrich Chemical Co. and were used without purification.

General procedures for compound synthesis
To a solution of an amine (1 eq.) and pyridine (4 eq.) in DMF (2 mL) was added chloroacetyl chloride (1.3 eq.). After 30 min at rt, the reaction was added to a mixture of EtOAc and water (1:1). The aqueous layer was extracted with EtOAc and the organic extraction was washed with water. The organic extraction was concentrated under reduced pressure to yield the desired α-chloroacetamide.
A solution of the α-chloroacetamide (1 eq.), an amine (1 eq.) and cesium carbonate (1 eq.) in DMF was heated to 150°C for 5 min in a microwave reactor. The solution was filtered (0.45µm) and fractions were separated via reverse-phase HPLC in a gradient of MeCN in water (0.1%TFA). Fractions were combined and added to water:EtOAc (1:1) and added aq. NaHCO 3 .
The organic layer was collected and solvent was removed on air concentrator. Residue was resuspended in DCM/MeOH and filtered through phase separator into vial yielding the desired final products.

Lead optimization
Commercial 1,2,3,4-tetrahydroquinoline was treated with chloroacetyl chloride in the presence of pyridine to yield the 1. Next, the appropriate nitrogen heterocycle was reacted in a microwave reactor with 1 under basic conditions yielding the desired compounds. The SAR is outlined in Tables S1-S2. Our first library was designed to keep the right-hand dihydroquinoline constant and evaluate the left-hand heterocyclic portion (Table S2). If the six-membered ring was aromatized, the compound lost activity against AnKir1, 2. Addition of a carbonyl in the 4position of the tetrahydroindazole, 3, also led to an inactive compound. Interestingly, deletion of a nitrogen from 3 brought some activity back into 4 (12.1 mM). One compound (VU730, 5) retained its activity toward AnKir1 (IC 50 =2.4 mM), but lost activity toward Kir2.1 (IC 50 >30 mM). Expanding the ring system to incorporate a tetrahydroquinoline retained some activity against AnKir1 (6, 8.0 mM), and deletion of the 6-membered ring of the tetrahydroisoquinoline leaving the unsubstituted pyrazole was unproductive (7, inactive). Our next library kept the left-hand trifluoromethyl tetrahydropyrazole (VU041, 1) constant while altering the right-hand amide portion of the molecule (Table S3).
Moving from the tetrahydroquinoline to the tetrahydroisoquinoline led to an ~7-fold loss of potency (8, 15 mM). Moving to the decahydroquinoline (9, 6.7 mM) retained some activity while the regioisomer was inactive (10).
Moving to the piperidine ring was not productive (11); however, adding pendant substitution led to active compounds (phenyl, 12, 5.6 mM and dimethyl, 13, 4.5 mM).   The pH of all solutions was adjusted to 7.5 with NMDG-OH.