Journal: bioRxiv

Article Title: Prioritizing Neuroactive Ligands Using Motif-Guided Virtual Discovery and Zebrafish Profiling

doi: 10.64898/2026.01.15.699747

Figure Lengend Snippet: ( A ) PRESTO-Tango GPCR assay results of HCRTR2 antagonists. Relative luminescence values were normalized against a positive control of OxA. Drugs are separated by a vertical dashed line, with samples with significant differences from the OxA control (p < 0.05) on the left and non-significant samples on the right. Significance determined using a one-tailed Welch’s t-test ( B ) Rosetta-predicted interaction energies (Rosetta Energy Units, REU) for contacts in the binding pocket of the seven strongest antagonists (black), suvorexant (magenta), and TAK-925 (green). ( C ) Binding mode of three antagonists with GLN134 in magenta and ASN324 blue. Arrows on 5262 indicate interactions that were eliminated with analogs. Pymol sessions are available on the linked Zenodo repository. ( D ) PRESTO-Tango dose-response curves for corresponding antagonists 6566 ( IC 50 = 5.78e-7 M), 8519 ( IC 50 = 6.13e-7 M), and 5262 ( IC 50 = 4.811e-8 M). ( E ) Jaccard similarity of residue interaction profiles between 7 strongest experimental antagonists (black), published antagonists (magenta), and published agonists (green). ( F ) PRESTO-Tango GPCR assay results for experimental antagonist 5262 and two analog drugs with modified functional groups to inhibit predicted residue interactions. * = p < 0.05 for analog vs 5262, # = p < 0.01 for 5262 and DMSO vs OxA (one-tailed Welch’s t-test). ( G ) Presto Tango GPCR assay results for four weak HCRTR2 agonists at 1e-4 M with percentage RLU compared to TAK-861 at 1e-5 M. Groups significantly different to DMSO using a one-tailed Welch’s t-test are labeled with * (p < 0.05) or ** (p < 0.01).

Article Snippet: Cells were incubated for 24 hours at 37°C, followed by transfection with a cocktail of the PRESTO-Tango HCRTR2 construct (Addgene plasmid # 66399), serum-free Opti-MEM (Gibco), and FuGENE (Promega).

Techniques: Positive Control, Control, One-tailed Test, Binding Assay, Residue, Modification, Functional Assay, Labeling

Journal: bioRxiv

Article Title: Prioritizing Neuroactive Ligands Using Motif-Guided Virtual Discovery and Zebrafish Profiling

doi: 10.64898/2026.01.15.699747

Figure Lengend Snippet: ( A ) Summary of zebrafish activity on the first experiment day (9:00-23:00, 5 dpf) following heat shock, comparing drug-treated (20 μM) to DMSO-treated larvae. The number of bouts was normalized against the average bout count from the control group per experiment to between experiments. Although non-significant in this summary plot, the 6566 compound displays significant antagonism when assessed with a linear mixed model ( Supplementary Fig 8 ). ( B ) Example multi-day activity profile of larval zebrafish following heat shock for a functional antagonist. The zebrafish experience heat shock on the afternoon of 4 dpf and the activity tracking begins that night. This timecourse includes the same day 1 data as is summarized per fish in panel A. ( C ) Example multi-day activity profile of hcrtr2 mutant larval zebrafish, comparing drug-treated (5 μM, as suvorexant is lethal at higher doses) to DMSO-treated. ( D ) Summary of hcrtr2 mutant larval zebrafish activity on the first experiment day (9:00-23:00, 5 dpf), comparing drug (20 μM, except suvorexant, which was 5 μM) to DMSO. For improved visualization, data for 5682 is available only in Supplementary Fig. 8 . ( E ) Percent binding pocket residue identity (blue) and similarity (orange) determined for 210 GPCRs. Human and zebrafish systems were aligned against each other, and the closest residue, if any, within 2 angstroms was considered for comparison. If no residue was found within two angstroms, the residue comparison was not accounted for in the identity or similarity determination.

Article Snippet: Cells were incubated for 24 hours at 37°C, followed by transfection with a cocktail of the PRESTO-Tango HCRTR2 construct (Addgene plasmid # 66399), serum-free Opti-MEM (Gibco), and FuGENE (Promega).

Techniques: Activity Assay, Control, Functional Assay, Mutagenesis, Binding Assay, Residue, Comparison