Authors :
Presenting Author: Mustafa Hameed, MD – Boston Children's Hospital
Yongho Choe, BA – Boston Children's Hospital
Jed Hubbs, PhD – Boston Children's Hospital
Bruce Bean, PhD – Harvard Medical School
Alexander Rotenberg, MD, PhD – Boston Children's Hospital, Harvard Medical School, Boston, MA, USA
Rationale:
Rapid parvalbumin-positive interneuron (PVI) firing is critical for maintaining cortical GABAergic inhibitory tone. The Kv3.1 channel enables rapid PVI axonal repolarization and a shortening of the spike width, which is necessary for these neurons’ fast spiking physiology. We previously identified that Kv3.1 protein expression is decreased in mice who experience repetitive convulsive seizures, and also demonstrated seizure suppression by AUT1, a commercially available tool Kv3.1 positive allosteric modulator (PAM). Since Kv3.1 PAMs are unavailable for use in humans, we aim to develop novel compounds to activate Kv3.1 for purposes of seizure suppression and, perhaps, for epilepsy prevention.
Methods: Ten novel analogs of published compounds were designed (new chemical entities, NCEs). NCEs were compared to AUT1
in-vitro for Kv3.1 activity on an automated patch clamp system using plated Chinese hamster ovary (CHO) cells expressing cloned human Kv3.1b channels (n=4-12 wells/condition). Potassium current was measured at -20 mV during a stair-step voltage protocol, baseline at -80 mV subtracted. Lead compounds were tested in a p17-19 Scn1a haploinsufficient (Scn1a+/-) mouse febrile seizure (FS) model.
Results: NCE-049 activated Kv3.1b channels more effectively than AUT1 at both 3 uM (relative stair current; AUT1: 0.98 ± 0.51; NCE-049: 1.44 ± 0.95) and 10 uM (AUT1: 1.93 ± 1.07; NCE-049: 3.12 ± 2.08). Next-best Kv3.1 activation was by NCE-052 (3 uM: 1.12 ± 0.31; 10 uM: 1.55 ± 0.53). Given compound availability, we tested NCE-052 in the mouse FS model (vehicle: N = 7; NCE-052: N = 8). NCE-052 treatment delayed seizure onset (log-rank test; p< 0.01; Figure 1A) and significantly increased mean FS temperature threshold (increase from baseline temperature; VEH: +5.2 ± 2.1C; NCE-052: +8.7 ± 1.6C; unpaired t-test; p< 0.01; Figure 1B). None of the NCE-052 treated mice developed FS at the VEH group median FS threshold of +4.7C from baseline (fisher’s exact test; p< 0.05; Figure 1C).