PRECLINICAL EVIDENCE SUPPORTING THE ROLE OF ALOGABAT, A GABAA-α5 POSITIVE ALLOSTERIC MODULATOR, AS A NOVEL THERAPEUTIC APPROACH FOR NEURODEVELOPMENTAL DISORDERS

Abstract Background There is compelling evidence that dysfunction of the GABAergic system, the main inhibitory neurotransmitter system in the brain, contributes to the pathophysiology of neurodevelopmental disorders including autism spectrum disorder and deletion Angelman syndrome (1, 2, 3, 4). Genetic studies highlight the critical role of GABAA receptor gene dosage and function within the 15q11-13 chromosomal region in the etiology of these conditions (5, 6, 7, 8). This region encompasses several genes including UBE3A and GABRB3, GABRA5, GABRG3 encoding the β3, α5, and g3 GABAA receptor subunits, respectively. These subunits together with the g2 subunit co-assemble to form the GABAA- α5 receptor subtype (9). Therefore, positive modulation of GABAA-α5 receptors may provide a novel therapeutic approach to restore deficient GABAergic signaling without the typical side effects of non- selective GABAA positive allosteric modulators (PAM) i.e., diazepam. Aims & Objectives Assess the in vitro and in vivo pharmacological profile of alogabat, a novel small molecule, selective GABAA-α5 receptor positive allosteric modulator. Provide preclinical proof of concept data to support clinical studies in ASD and Angelman syndrome. Methods Alogabat was evaluated for selective binding and functional activity in vitro at GABAA-α5β3g2 receptors, including electrophysiological studies in hippocampal slices. In vivo studies included receptor occupancy (RO) using a selective GABAA-α5 tracer (autoradiography), pharmacological magnetic resonance imaging (phMRI) and EEG in rodents. Alogabat was assessed on the repetitive behavior phenotype in BTBR and contactin-associated protein-like 2 knockout (Cntnap2-/-) mice, on seizure models and cognitive performance in rats, and on rotarod performance following a combination treatment with diazepam. Results Alogabat is a potent PAM of the GABAA-α5 receptor with binding and functional selectivity. RO studies provided direct proof of dose-dependent target engagement. Functional circuit modulation was demonstrated by dose-dependent regional perfusion changes in phMRI and change in EEG theta and beta band power. Alogabat at >50% RO at GABAA-α5 normalized elevated self-grooming in both Cntnap2-/- and BTBR mice and exhibited antiepileptic activity in rats. Alogabat did not impair cognition in rats at RO up to 88%, although impairment occurred at higher doses probably due to decreased α-subunit selectivity. Alogabat did not worsen diazepam-induced rotarod impairment. Conclusions Alogabat showed beneficial effects in mouse models relevant for NDD, as well as anti- seizure activity, at doses without cognitive, sedative, and motoric side effects. In addition, translational biomarkers were identified to guide clinical studies: PET, phMRI, EEG. References 1. Braat S, Kooy RF. The GABAA receptor as a therapeutic target for neurodevelopmental disorders. Neuron 2015;86:1119-30. 2. Ali Rodriguez R, Joya C, Hines RM. Ribs of inhibitory synaptic dysfunction in the umbrella of neurodevelopmental disorders. Front Mol Neurosci. 2018;11:132. 3. Tang X, Jaenisch R, Sur M. The role of GABAergic signalling in neurodevelopmental disorders. Nature Reviews Neuroscience. 2021 (5):290-307. 4. Zhao H, Mao X, Zhu C, et al. GABAergic system dysfunction in autism spectrum disorders. Front Cell Dev Biol. 2022; 9:781327. 5. Warrier V, Baron-Cohen S, Chakrabarti B. Genetic variation in GABRB3 is associated with Asperger syndrome and multiple endophenotypes relevant to autism. Mol Autism 2013;4:48. 6. Zurek AA, Kemp SW, Aga Z, et al. a5GABAA receptor deficiency causes autism-like behaviors. Ann Clin Transl Neurol 2016;3:392-8. 7. Hogart A, Wu D, LaSalle JM, et al. The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol Dis 2010;38:181-91. 8. Frohlich J, Miller MT, Bird LM, et al. Electrophysiological phenotype in Angelman syndrome differs between genotypes. Biol Psychiatry 2019; 85(9):752-9. 9. Sur C, Fresu L, Howell O, et al. Autoradiographic localization of alpha5 subunit-containing GABAA receptors in rat brain. Brain Res 1999; 822:265-70.