Table 1.
Compositions (in mM) of solutions used in Xenopus oocyte electrophysiology.
Figure 1.
Tl+ flux assay of AeKir1 channel activity for high-throughput screening.
(A) Representative Tl+-induced changes in Thallos fluorescence in T-Rex-HEK293-AeKir1 cells cultured overnight with (+Tet) or without (-Tet) tetracycline. The shaded box indicates the cell exposure to Tl+. (B) DMSO concentrations up to 1.3% v/v DMSO have no effect on Tl+ flux through AeKir1. Data are means ±SEM (n = 3). One-way ANOVA P<0.0001, and asterisks (**, ***) indicate P<0.01 or P<0.001 respectively, when compared to 0% DMSO (Tukey's test). (C) Representative checkerboard analysis using 100 µM VU573 or 0.1% v/v DMSO as the vehicle control. The mean peak fluorescence amplitude of each sample population is indicated with a solid line and alternating samples for DMSO (top) and VU573 (bottom) are graphed as individual points. The mean ±SD Z′ calculated over 6 plates on 3 separate days was 0.69±0.05.
Figure 2.
VU625 is a potent inhibitor of AeKir1 in Tl+ flux assays.
(A) Chemical structure of VU625. (B) Dose-dependent inhibition of the AeKir1-mediated Tl+ flux by VU625 with concentrations ranging from ≤0.12 to 30 µM. The arrow indicates when Tl+ was added to the extracellular bath. (C) Concentration-response curves of VU625 derived from Tl+ flux assays. The IC50 and Hill-coefficient (nH) values are 315 nM (95% CI: 254.4–390.2 nM) and 0.98 respectively. Data are mean ±SEM. n = 4 independent experiments performed in triplicate.
Figure 3.
VU625 is a potent and preferential inhibitor of AeKir1 over AeKir2B in whole-cell electrophysiology.
(A) Normalized AeKir1 current-voltage relationships obtained from heterologous expression in T-Rex-HEK293 cells, illustrating VU625-dependent inhibition before (control) and after addition of 0.9 µM VU625. Residual AeKir1 currents were inhibited with 2 mM barium. Cells were voltage clamped at −75 mV and ramped between −120 mV and +60 mV. (B) Concentration-response curve of VU625 derived from patch clamp experiments (n = 4–6). The IC50 of VU625 is 96.8 nM (95% CI: 75.4–124.2 nM). (C) Concentration-response curves of current inhibition mediated by heterologous expression in Xenopus oocytes of AeKir1 (filled circles) and AeKir2B (open circles) channels after bath application of VU625. n = 4–5 oocytes per concentration. The calculated IC50 values of VU625 for AeKir1 and AeKir2B current inhibition are 3.8 µM (95% CI: 2.3–6.3 µM) and 45.1 µM (95% CI: 31.7–64.2 µM), respectively.
Figure 4.
Design and chemical lead optimization strategy for VU625.
(A) Modular approach to assess three areas of diversification of VU625: sulfonamide (red shading), central core (green shading), and southern amide (blue shading) portions. (B) General synthetic approach to access VU625 and analogs around the amide and sulfonamide portions.
Table 2.
Structure-activity relationships and lead optimization summary of VU0077625 scaffold.
Table 3.
Structure-activity relationships and lead optimization summary for the central core portion of VU0077625 scaffold.
Figure 5.
Summary of structure-activity relationship (SAR).
Summary of observed SAR of over 100 analogs synthesized exploring all three regions of VU625.
Figure 6.
Effects of probenecid and VU625 on survival of adult female mosquitoes (A. aegypti).
Percent mortality of mosquitoes at 24 h post-injection. Each mosquito was injected with 69 nl of the vehicle containing VU625 (10 mM), probenecid (50 mM), or both, to deliver the desired doses: 690 pmol of VU625, 3.4 nmol probenecid. n = 6–7 trials of 10 mosquitoes each per treatment. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA with a Newman-Keuls post-test (P<0.05).
Figure 7.
The dose-response curve of the toxic effects of VU625 on adult female mosquitoes (A. aegypti) is biphasic.
Normalized percent mortality of mosquitoes at 24 h post-injection. Each mosquito was injected with 69 nL of the vehicle containing probenecid (50 mM) and an appropriate concentration of VU625 to deliver the doses of VU625 indicated and 3.4 nmol of probenecid. The ED25 and ED75 were determined by fitting a non-linear biphasic curve to the data. n = 3–4 trials of 10 mosquitoes each per dose.
Figure 8.
Effects of probenecid and VU625 on the in vivo excretory capacity of adult female mosquitoes (A. aegypti).
Amount of urine excreted by mosquitoes 1 h after injection with 900 nL of the vehicle (K+-PBS50 containing 1.8% DMSO, 0.077% β-cyclodextrin, and 0.008% Solutol), or the vehicle containing VU625 (0.77 mM), probenecid (3.85 mM), or both, to deliver the desired doses: 690 pmol of VU625, 3.4 nmol probenecid. Values are means ±SEM; n = 6–18 trials of 5 mosquitoes per treatment. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA with a Newman-Keuls posttest (P<0.05).