Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1/SUR2B KATP Channels

Abstract

Vascular smooth muscle K_ATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular K_ATP) over Kir6.2/SUR1 (pancreatic K_ATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC₅₀ of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific K_ATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific K_ATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle K_ATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific K_ATP channel inhibitor, VU0542270.

Graphical abstract

Fig. 1.

High-throughput screening assay development for Kir6.1/SUR2B. (A) Cartoon depiction of the thallium flux assay used for HTS. Kir6.1 (purple)/SUR2B (blue) channels expressed in HEK-293 cells are opened with pinacidil (yellow triangle) before adding extracellular thallium (Tl⁺) to induce flux through the pore and detection with Thallos Brilliant dye (green circles). (B) CRC data showing activation of Kir6.1/SUR2B, but not Kir6.2/SUR1, with pinacidil (n = 9 at each dose). (C) CRC data showing opening of Kir6.2/SUR1, but not Kir6.1/SUR2B, with VU0071063. (D) Sensitivity of thallium assay to increasing doses of DMSO. Individual data points with means ± S.D. (n = 9 at each dose) are shown. (E) Pseudo-colored fluorescence image of 384-well plate containing HEK-293-Kir6.1/SUR2B cells treated with 10 µM pinacidil (green wells) or 10 µM pinacidil + 10 µM glibenclamide (blue wells) approximately 30 seconds after thallium addition. (F) Scatter plot of fluorescence values measured from the plate in E1 showing clear separation of the twowell populations (i.e., pinacidil versus pinacidil plus glibenclamide). 0% inhibition is defined as cell treated with 10 µM pinacidil, and 100% inhibition is defined as cell treated with 10 µM pinacidil + 10 µM glibenclamide. The calculated Z’ for this plate was 0.64.

Fig. 2.

Discovery of VU0542270 in a screen of 47,872 compounds. (A) Summary of screening results. Wells were treated with 10 µM pinacidil (negative control; 0% inhibition), 10 µM pinacidil + 10 µM glibenclamide (positive control; 100% inhibition), or 10 µM pinacidil + test compounds. Each point represents the value of a single well of a 384-well plate using the slope normalized to maximal inhibition (i.e., pinacidil plus glibenclamide). Inhibitors are defined as those decreasing thallium-induced fluorescence by 3 S.D. below the mean pinacidil response and 3 mean absolute deviation below the median pinacidil plate response. Six hundred thirty-nine inhibitors (black) and 44,267 inactives are shown. Not shown are putative activators, fluorescent compounds, and retest negatives (2966). (B) Chemical structure of VU0542270. (C) Representative fluorescence traces from single wells treated with pinacidil (blue), pinacidil plus glibenclamide (red), or pinacidil plus VU0542270 (black).

Fig. 3.

Electrophysiological characterization of VU0542270-dependent inhibition of Kir6.1/SUR2B. (A) Representative whole-cell currents recorded from HEK-293-Kir6.1/SUR2B cells bathed in control buffer (top, left), 1 µM pinacidil (top, right), 1 µM pinacidil + 10 µM VU0542270 (bottom, left), or 1 µM pinacidil + 10 µM glibenclamide (bottom, right). Cells were voltage clamped at a holding potential of −75 mV and stepped between −120 Mv and +120 mV in 20-mV increments. Current amplitude has been normalized to cell capacitance (pA/pF). (B) Current-voltage relationships measured under the indicated conditions (n = 4–6). (C) Representative time-course of Kir6.1/SUR2B channel activation by pinacidil (1 µM) and inhibition by the indicated dose of VU0542270 or glibenclamide (10 µM). (D) CRC data showing Kir6.1/SUR2B-dependent inhibition by VU0542270 (n = 4–6 at each dose). Fitting a four-parameter logistic function to the data yields an IC₅₀ = 278 nM.

Fig. 4.

VU0542270 selectivity is mediated through SUR2. HEK-293 cells were transfected with plasmids encoding (A) Kir6.2/SUR1 (VU0071063 activation), (B) Kir6.2/SUR1 (ATP depletion), or (C) Kir6.1/SUR2B (pinacidil activation), treated with escalating doses of glibenclamide (circles) or VU0542270 (squares), and evaluated in thallium flux assays. Data are individual data (n = 9 wells/dose) fitted with four-parameter logistic functions to derive IC₅₀ values as follows: (A) glibenclamide = 12 nM, VU0542270 = no fit, (B) glibenclamide = 10 nM, VU0542270 = no fit, (C) glibenclamide = 115 nM, and VU0542270 = 129 nM. (D) Mean ± S.D. per cent inhibition of the indicated Kir6/SUR combination with 3 µM VU0542270 (n = 9 wells/dose). M, molar.

Fig. 5.

Synthesis of VU0542270 and initial SAR study plan. (A) The synthetic route for VU0542270. (Ai) LAH, THF, 0°C; (Aii) SOCl₂, CHCl₃, 0°C; (Aiii) NaN₃, K₂CO₃, DMF, 70°C; (Aiv) PPh₃, EtOAc, THF, 50°C. (B) Initial two-track SAR study approaches; iterative parallel synthesis and SAR by catalog. (C) Selected compounds from the initial SAR study.

Fig. 6.

VU0542270 induces constriction of isolated mouse DA vessels. Isolated DA vessel before (A) and after (B) exposure to high extracellular potassium (i.e., 50 mM bath KCl), demonstrating vascular reactivity to membrane depolarization. (C) Vasoconstriction of isolated DA vessels in response to escalating doses of glibenclamide alone (black circles) or together with pinacidil (open circles). (D) Vasoconstriction of isolated DA vessels in response to escalating doses of VU0542270 alone (closed circles) or together with 10 µM pinacidil (open circles). Data are means ± S.D. from 4–6 vessels. M, molar.