Macrolides selectively inhibit mutant KCNJ5 potassium channels that cause aldosterone-producing adenoma

Abstract

Aldosterone-producing adenomas (APAs) are benign tumors of the adrenal gland that constitutively produce the salt-retaining steroid hormone aldosterone and cause millions of cases of severe hypertension worldwide. Either of 2 somatic mutations in the potassium channel KCNJ5 (G151R and L168R, hereafter referred to as KCNJ5MUT) in adrenocortical cells account for half of APAs worldwide. These mutations alter channel selectivity to allow abnormal Na+ conductance, resulting in membrane depolarization, calcium influx, aldosterone production, and cell proliferation. Because APA diagnosis requires a difficult invasive procedure, patients often remain undiagnosed and inadequately treated. Inhibitors of KCNJ5MUT could allow noninvasive diagnosis and therapy of APAs carrying KCNJ5 mutations. Here, we developed a high-throughput screen for rescue of KCNJ5MUT-induced lethality and identified a series of macrolide antibiotics, including roxithromycin, that potently inhibit KCNJ5MUT, but not KCNJ5WT. Electrophysiology demonstrated direct KCNJ5MUT inhibition. In human aldosterone-producing adrenocortical cancer cell lines, roxithromycin inhibited KCNJ5MUT-induced induction of CYP11B2 (encoding aldosterone synthase) expression and aldosterone production. Further exploration of macrolides showed that KCNJ5MUT was similarly selectively inhibited by idremcinal, a macrolide motilin receptor agonist, and by synthesized macrolide derivatives lacking antibiotic or motilide activity. Macrolide-derived selective KCNJ5MUT inhibitors thus have the potential to advance the diagnosis and treatment of APAs harboring KCNJ5MUT.

Figure 1. Establishment of screening assays for KCNJ5^MUT and KCNJ5^WT inhibition.

(A) Reduced ATP levels following induction of KCNJ5^G151R or KCNJ5^L168R, but not KCNJ5^WT, in HEK293 cells. Cell survival was measured using an ATP-based viability assay with luminescence as a read-out in stable inducible cell lines in the uninduced and induced states. Whereas induction of KCNJ5^WT expression had no significant effect on ATP levels, induction of KCNJ5^G151R and KCNJ5^L168R led to a significant decrease in ATP levels. P values represent 2-tailed unpaired Student’s t test comparing uninduced vs. induced for each line. n = 8 for each condition, 2,000 cells per well (WT and G151R) or 4,000 cells per well (L168R). Bars represent mean, and error bars represent SD. (B) Evaluation of the KCNJ5^WT membrane potential assay using BaCl₂ using a standard curve of BaCl₂ concentrations. Depolarization caused by increasing BaCl₂ concentrations leads to an increase in fluorescence. Relative fluorescence at 2 mM BaCl₂ was defined as 100%. Data were fitted with a 4-parameter nonlinear regression of log-dose versus response. n = 6. Dots represent mean, error bars represent SD.

Figure 2. Structure activity relationship of macrolide inhibition of KCNJ5^G151R.

Select compounds are shown. The locations of desosamine and cladinose moieties are indicated on the roxithromycin structure. Values of IC₅₀ against KCNJ5^G151R are given for active compounds. See Table 1 for detailed screen results.

Figure 3. Dose-response curves of lead macrolide compounds on WT and mutant KCNJ5.

Dose-response curves of roxithromycin, clarithromycin, and idremcinal on activity of WT and mutant (G151R and L168R) KCNJ5 are shown. For mutant KCNJ5, percentage of inhibition denotes increase in ATP, measured by luminescence, in the presence of drug. For WT KCNJ5, percentage of inhibition denotes change in membrane potential in the presence of drug (see Methods). Compounds were tested in technical triplicates at 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.0781, and 0.0391 μM concentrations. Data were fit with a 4-parameter nonlinear regression of log-dose versus response. While inhibition of mutant channels showed a strong dose-response relationship with all 3 compounds, there was no significant inhibition of WT channels. See Table 1 for inhibition data.

Figure 4. Structures of synthesized compounds with and without inhibition of mutant KCNJ5.

Values of IC₅₀ for KCNJ5^G151R are given for active compounds. See Supplemental Table 3 for detailed screen results.

Figure 5. Roxithromycin inhibits KCNJ3/KCNJ5^MUT-induced Na⁺ current and KCNJ5^MUT-induced aldosterone production.

(A) Cation currents were measured with the perforated whole cell recording (see Methods). Current-voltage plots of KCNJ3/KCNJ5^G151R, KCNJ3/KCNJ5^L168R, and KCNJ3/KCNJ5^WT heterotetramers before and 10 minutes after addition of 20 μM roxithromycin and 1 mM BaCl₂ are shown. Shown are 3 (G151R, WT) or 4 (L168R) biological replicates. (B) HAC15 cells stably transfected with KCNJ5^WT, KCNJ5^G151R, KCNJ5^L168R, or empty vector were treated with the indicated concentrations of roxithromycin or with vehicle control for 6 hours. Aldosterone in the supernatant was quantified by ELISA and normalized to cell count (see Methods). Four independent experiments were performed for each condition (each measured in duplicate of 2 dilutions). (C) H295R cells transiently transfected with KCNJ5^WT, KCNJ5^G151R, KCNJ5^L168R, or empty vector were treated with roxithromycin or vehicle control for 18 hours. CYP11B2 expression (with GAPDH as control) was determined by qPCR; 6 independent experiments were performed. Values were normalized to the respective values of empty vector. Error bars represent SEM. NS denotes P > 0.05. *P ≤ 0.05; **P ≤ 0.01; ^#P ≤ 0.001, Mann-Whitney U test versus 0 μM roxithromycin.

Figure 6. Inhibition of KCNJ5^MUT by PLUX37 and PLUX38.

Cation currents of heterotetramers of KCNJ3 and mutant or WT KCNJ5 were measured with the perforated patch-clamp technique without (blue) or with (red) 10 μM inhibitor (A) PLUX37 and (B) PLUX38. Extracellular solution: 140 mM NaCl/5 mM KCl; intracellular solution: 140 mM KCl. For both compounds, n = 5 biological replicates for KCNJ3/KCNJ5^G151R and KCNJ3/KCNJ5^L168R; n = 4 for KCNJ3/KCNJ5^WT (mean and SEM). (C) H295R cells transiently transfected with KCNJ5^WT, KCNJ5^G151R, KCNJ5^L168R, or empty vector were treated with PLUX38 or vehicle control for 18 hours. CYP11B2 expression (with GAPDH as control) was determined by qPCR; 5 independent experiments were performed. Values were normalized to the respective values of empty vector. Error bars represent SEM. NS denotes P > 0.05. **P ≤ 0.01, Mann-Whitney U test versus 0 μM roxithromycin.