Exploring the ligand-binding potential of the Neuronal PAS proteins NPAS1 and NPAS3, and their partner ARNT2

Thesis

Mammalian basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factors play key roles in various human diseases and are emerging as promising drug targets due to their ability to bind small molecules within their PAS domains. Among them, the neuronal PAS proteins NPAS1 and NPAS3, together with their heterodimerisation partner ARNT2, have been linked to neurological conditions, notably schizophrenia, autism spectrum disorders, intellectual disabilities, and bipolar disorder, making them promising candidates for therapeutic targeting.

This DPhil project identifies novel small molecules for NPAS1, NPAS3, and their partner, ARNT2. A high-throughput biochemical screen comprising 67,847 compounds was conducted with NPAS1-ARNT2, NPAS3-ARNT2, and ARNT2-PAS-B proteins. Reconfirmed hits were evaluated for binding affinity using MST and SPR. Crystal structures of ligand-bound ARNT2-PAS-B revealed that compounds occupy a large cavity within the domain, and interact with His341, a residue critical for ligand stabilisation. Ligand binding induced conformational changes that enlarged the binding pocket, and disrupted interfaces critical for heterodimer stability.

Hit-to-lead optimisation using the known ARNT2 binder KG-548, and butein, yielded ligand series designed to reduce PAINS liabilities and improve affinity, metabolic stability, and selectivity for the ARNT2 PAS-B domain. Distinct yet complementary binding modes of these compounds highlighted the PAS-B pocket’s flexibility in accommodating diverse scaffolds through hydrogen bonding, ionic interactions, and water-mediated networks.

This study identifies ligands that directly target the ARNT2 PAS-B domain, and demonstrates their ability to modulate heterodimer stability through structural and biochemical characterisation. Compounds 3617-M16, FR-15, FR-20, and FR-26 emerge as promising probes, which may be used for exploring the functional role of ARNT2. These findings provide a framework for designing next-generation ARNT2 modulators and for investigating ligand-dependent regulation of NPAS-ARNT2 transcriptional activity in health and disease.

Authors: Bazylianska, V

• DOI: 10.5287/ora-qy46pogq6
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: KG-548; bHLH–PAS family; drug discovery; butein; protein–ligand interaction; NPAS3; ARNT2; PAS domain; transcription factor; NPAS1
• Subjects: Drug Discovery; Clinical medicine; Biochemistry; Biology