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.