Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-d]oxazol-2-amine (SKA-121).

Intermediate-conductance (KCa3.1) and small-conductance (KCa2) calcium-activated K+ channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing KCa activators, such as naphtho[1,2-d]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potencies. In a previous chemistry effort, we optimized the benzothiazole pharmacophore of SKA-31 toward KCa3.1 selectivity and identified 5-methylnaphtho[2,1-d]oxazol-2-amine (SKA-121), which exhibits 40-fold selectivity for KCa3.1 over KCa2.3. To understand why introduction of a single CH3 group in five-position of the benzothiazole/oxazole system could achieve such a gain in selectivity for KCa3.1 over KCa2.3, we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used computational modeling software to generate models of the KCa3.1 and KCa2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling, we suggest that all benzothiazole/oxazole-type KCa activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen bond with E54 on CaM. In KCa3.1, SKA-121 forms an additional hydrogen bond network with R362. In contrast, NS309 sits more "forward" and directly hydrogen bonds with R362 in KCa3.1. Mutating R362 to serine, the corresponding residue in KCa2.3 reduces the potency of SKA-121 by 7-fold, suggesting that R362 is responsible for the generally greater potency of KCa activators on KCa3.1. The increase in SKA-121's KCa3.1 selectivity compared with its parent, SKA-31, seems to be due to better overall shape complementarity and hydrophobic interactions with S372 and M368 on KCa3.1 and M72 on CaM at the KCa3.1-CaM-BD/CaM interface.