Discovery of CVN293, a Brain Permeable KCNK13 (THIK-1) Inhibitor Suitable for Clinical Assessment

Abstract

The potassium (K⁺) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1β from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.

Figure 1

Previously reported KCNK13 inhibitors.

Figure 2

Single crystal X-ray structure confirming the intramolecular hydrogen bond in CVN293.

Figure 3

Inhibitory activity of CVN293 for the human and mouse forms of KCNK13, KCNK2, and KCNK6. CVN293 inhibitory activity was determined in HEK-293 cells expressing KCNK13, KCNK2, or KCNK6 in thallium flux assays. Data shown represent the averaged responses (initial slope; ± SD) normalized to controls from two independent experiments, each performed in duplicate.

Figure 4

CVN293 prevents IL-1β release from murine microglia. Neonatal microglia cultures were primed with LPS and activated by the removal of extracellular K⁺ (A) following the treatment with CVN293. Data are presented as a percentage of DMSO-treated cells and represent the mean ± SEM of five independent experiments. Data represent the mean ± SEM of five independent experiments. Statistical difference was calculated by two-way ANOVA followed by a multiple comparison with Tukey’s model. Hippocampal slice cultures were primed with LPS with or without activation with ATP (5 mM) following the treatment with CVN293 (1 μM; B and C). Data represent the mean ± SEM of at least five independent experiments. Statistical difference was calculated by one-way ANOVA followed by Dunnett’s posthoc analysis. **P < 0.01.

Scheme 1. (a) EtOH, 80 °C, 40%; (b) K₂CO₃, DMF, RT, 25% 9, 20% 12; (c) Et₃N, CH₃CN, 90 °C, 70%; (d) Fe⁰, NH₄Cl, EtOH, H₂O, 100 °C, 89%; (e) 22, EtOH, 90 °C, 10%