Compound screening in sensory neurons
We previously reported that targeting the Cav2.2–CRMP2 interaction with tat-CBD3 (Brittain et al., 2011b) or myr-tat-CBD3 (Francois-Moutal et al., 2015) peptides results in reduction of depolarization-induced Ca2+-influx in sensory neurons. Here, we used this approach for experimental validation. For this, we used Fura 2-AM-based ratiometric Ca2+-imaging in rat DRG neurons. To activate high-voltage-activated (HVA) Ca2+ channels, we challenged DRG neurons from all sizes with 90 mM KCl (Gomez et al., 2022). We have previously demonstrated that acute application (5-30 minutes) of 20 µM of myr-tat-CBD3 peptide was unable to inhibit Ca2+influx. In contrast, chronic application (~24 hours) of 20 µM of myr-tat-CBD3 peptide inhibited Ca2+ currents by ~40% (Francois-Moutal et al., 2015). Hence, overnight incubation of 20 µM of test compounds was utilized to assess the activity of these compounds.
Our in vitro screening showed that stimulation of DRG neurons with 90 mM KCl led to an increase in Ca2+ influx as shown in the control group (0.05% DMSO; Figure 2A ). Overnight incubation with 20 μM of the test compounds revealed that, of the 77 compounds tested, nine of them (CBD3018, 3026, 3033, 3038, 3039, 3062, 3063, 3065 and 3074) inhibited Ca2+ influx by more than 50% relative to the DMSO-treated (i.e., control) group (Figure 2A ). With one exception (CBD3026), all identified antagonists contained similar chemotypes – a protonated moiety and two dimethylamines, as well as the alanine hydrophobic moiety (Figure S3 ). Indeed, all nine compounds can be assigned to only two chemical classes and three chemotypes. Specifically, CBD3018, 3026, 3062, 3065, 3074 belong to guanidines, CDB 3033, 3038, 3039 feature 2-aminopyridylpropylcarboxamide class, and CBD3062 and 3063 are of analogous 2-aminopyridylpropylurea chemotype. Overall, these results show that these first-in-class compounds predicted to disrupt Cav2.2–CRMP2 interaction decrease HVA channel activity in DRG neurons from all sizes and share chemically similar motifs.