(T1530-06-31) Discovery of Potassium Channel K_v7.4(KCNQ4) Selective Activator for the Treatment of Hearing loss

Purpose: Current treatments for hearing loss are directed at non-specific targets, such as inhibiting free radicals or inhibiting cell death mechanisms. We set out to find a specific target protein for hearing loss treatment and found Potassium Channel K_v7.4(KCNQ4), a protein that is highly affected by noise. KCNQ4 is a gene that encodes a potassium channel protein, which is crucial for regulating electrical signaling in cells. In the cochlea, it is expressed in hair cells and is involved in hair cell repolarization and homeostasis of K+ ions. When the amount of potassium ions entering the cell increases due to a weakened function of KCNQ4 or excessive noise exposure, K+ ions remain in high concentrations in the cell, causing the outer hair cell to undergo degenerative cell necrosis due to constant depolarization. In addition, if KCNQ4 is not functioning properly due to a mutation, K+ ions cannot be released, which can cause hearing loss. No drug has yet been developed that can specifically activate KCNQ4. Therefore, the development of a KCNQ4 activator will be an innovative new drug for the treatment of noise-induced hearing loss, and it can also serve as an important drug in the study of physiology and pathophysiology related to KCNQ4. In addition, it has high economic development value as it can replace a significant portion of the hearing aid or cochlear implant market currently used to treat hearing loss. Other KCNQ activators such as Retigabine exist, but there is a side effect of activating other isotypes due to the lack of isotype selectivity. For example, activation of KCNQ2 may cause epilepsy. It is necessary to develop a KCNQ4 selective activator for the treatment of hearing loss.

Methods: We built a cell-based High Throughput Screening (HTS) for KCNQ4 activator heat compound discovery. The activity of the compound was assessed using FluxOR assay, which was fluorescent when mutant KCNQ4 was expressed in the LN 215 cell line and then combined with thallium. Using the HTS, ~98,000 new compounds were searched to discover primary hits. A derivative was produced from the discovered active material to evaluate its activity, and the secondary hits that were the best in efficacy evaluation using patch-clamp discovered in KCNQ4 over-expression CHO cell. After selecting a hit compound, in order to improve the activity and properties of the compound, the chemical structure constituting the parent structure was arbitrarily classified and deformation for each part was designed. For each part, the type and position of substituent, heteroatom change, and functional deformation were designed to secure a derivative accordingly. We designed 7 steps scheme and synthesized compounds using SAR analysis to find lead compound. For compounds showing better activity than NC00074594(NC94), the activity evaluation was also performed on KCNQ2 to determine if there was selectivity for KCNQ4.

Results: First, 37 compounds were selected for primary hits. (Figure 1) From the primary hits, 393 derivatives were produced to evaluate their activity, and secondary hit GK-13 was discovered. Based on the chemical structure of GK-13, a compound library was constructed. Then B2961 with increased activity and increased selectivity for KCNQ4 through SAR was selected as tool compound. B2961 has an EC50 of 242nM in KCNQ4 and has selectivity that does not show activity in KCNQ1 and KCNQ2. (Figure 2) When treated with microsome for 30 minutes, it showed moderated metabolic stability of about 46.5% and low solubility of less than 1 ug/ml in five buffered solutions including 3DW. With maintaining core structure of GK-13, we modified other moiety and synthesized 140 compounds for better activity and properties than B2961. With the introduction of the amide group in the 7 position, substances with better activity than esters could be found. When nitrogen was introduced to 7-position instead of sulfur, there is no active for KCNQ4, also metabolic stability improves with the introduction of Oxygen, but there is a possibility of cytotoxicity. When the other group was introduced instead of the tert-butyl group in the 5 position, the activity was reduced. When the substituted phenyl group is introduced into position 3, the activity increases when the 4-fluorophenyl group came in. Considering that the para position hydrogen of the phenyl group is a weak position in the microsomal stability, the 4-fluorophenyl group is important in terms of activity and stability. After optimizing process, we discovered the lead compound A4924 with improved KCNQ4 activity and selectivity. (Figure3)

Conclusion: In order to find compounds that can selectively act on KCNQ4, we used HTS and FluxOR assay. As a result, we discovered 37 compounds and determined Gk-13 as a hit compound and B2961 as a tool compound through further verification. With maintaining the core sturcture of Gk-13, we synthesized compounds with different moieties and conducted activity evaluation. As a result, we found A4924 as a lead compound, which has high activity and selectivity for KCNQ4.

References: 1) C Kubisch, et al., KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell, 1999, 96(3), 437-446.
2) Thomas J. Jentsch, Neuronal KCNQ potassium channels:physislogy and role in disease, Nature Reviews Neuroscience, 2000, 1, 21-30

Figure1. Discovery of 37 KCNQ4 channel modulators (primary hits) and comparison of activities with NC00074594 (NC94) for KCNQ4.

Figure2. Dose response curve of B2961. (a) for KCNQ4 (b) for KCNQ2.

Figure3. Activity and properties of A4924. Through these result, A4924 was selected as lead compound.