Introduction
In the central and peripheral nervous systems, transmembrane
Cav2.2 (N-type) voltage-gated calcium channels are
expressed in the dorsal root ganglia (DRG) and spinal dorsal horn (SDH)
– two important sites for nociceptive transmission (Hoppanova &
Lacinova, 2022). Within the spinal cord, Cav2.2 channels
are localized in the central terminals of primary afferent fibers where
they mediate the release of excitatory neurotransmitters (Heinke, Balzer
& Sandkuhler, 2004). Knockout mouse models of Cav2.2
(Hatakeyama et al., 2001; Kim et al., 2001) demonstrated a key role for
these channels in nociceptive pathways: CaV2.2 deficient
mice show reduced response to mechanical stimuli in the von Frey test
and increased tail flick latency in response to radiant heat, indicating
altered spinal reflexes (Irwin, Houde, Bennett, Hendershot & Seevers,
1951); however, in the hot plate test, which is an assay of supraspinal
nociceptive integration (Giglio, Defino, da-Silva, de-Souza & Del Bel,
2006), pain responses are unaltered (Kim et al., 2001). That expression
and function of Cav2.2 channels increases following
nerve injury (Cizkova et al., 2002; Yang et al., 2018; Yu et al., 2019b)
demonstrates plasticity of this target for therapeutic intervention.
Three drugs targeting Cav2.2 channels are commercially
available for management of neuropathic pain conditions. Ziconotide
(Prialt®) – a synthetic version of the cone snail toxin ω-conotoxin
MVIIA – is a Cav2.2 channel blocker and was the first
non-opioid intrathecal analgesic approved by the US Food and Drug
Administration for the treatment of intractable chronic pain (Doggrell,
2004). Its use is hampered by its invasive route of administration,
narrow therapeutic window, and a panoply of side effects. Gabapentin
(Neurontin®) and Pregabalin (Lyrica®) – ligands of α2δ-1 auxiliary
subunit of Cav2.2 channels – alleviate chronic pain by
disrupting Cav2.2 –α2δ-1 interaction to prevent
Cav2.2 trafficking to the plasma membrane (Bauer,
Rahman, Tran-van-Minh, Lujan, Dickenson & Dolphin, 2010; Hendrich et
al., 2008; Sutton, Martin, Pinnock, Lee & Scott, 2002). Both
gabapentinoids have low efficacy and present with serious side effects
(Evoy, Peckham, Covvey & Tidgewell, 2021). Misuse of gabapentinoids has
led to an increase in overdose-related deaths between 2019 and 2020
(Kuehn, 2022). Therefore, there is a critical need to develop novel
medicines that effectively manage pain without producing negative side
effects.
Alternative approaches have been devised to regulate the functional
activity of Cav2.2 channels by targeting proteins that
interact with them. In this regard, we identified collapsin response
mediator protein 2 (CRMP2) as a regulator of Cav2.2
trafficking and function (Brittain, Piekarz, Wang, Kondo, Cummins &
Khanna, 2009; Chi et al., 2009; Khanna et al., 2019). CRMP2 is a
microtubule-binding protein that regulates neuronal polarity in
vitro (Goshima, Nakamura, Strittmatter & Strittmatter, 1995; Inagaki
et al., 2001). Importantly, CRMP2 interacts with Cav2.2
(Brittain, Piekarz, Wang, Kondo, Cummins & Khanna, 2009; Khanna,
Zougman & Stanley, 2007). Overexpression of CRMP2 leads to enhanced
Cav2.2 currents and surface expression (Brittain,
Piekarz, Wang, Kondo, Cummins & Khanna, 2009; Chi et al., 2009) and
enhanced neurotransmitter release (Brittain, Piekarz, Wang, Kondo,
Cummins & Khanna, 2009; Chi et al., 2009) in hippocampal (Brittain,
Piekarz, Wang, Kondo, Cummins & Khanna, 2009) and DRG neurons (Chi et
al., 2009). A 15-amino-acid peptide (designated CBD3, for calcium
channel binding domain 3) generated from CRMP2 interfered with the
Cav2.2-CRMP2 protein-protein interaction and decreased
calcium influx, transmitter release, and acute, inflammatory, and
neuropathic pain (Brittain et al., 2011b). Homology‐guided mutational
analysis of CBD3 revealed an antinociceptive core in the first six amino
acids with two residues (Ala1 and Arg4)
accounting for most of the binding affinity (Moutal et al., 2018).
In the absence of any significant structural information on
Cav2.2 to guide a rational design of small molecule
inhibitors, we developed a novel pipeline that leverages molecular
dynamics of the core peptide and identified the
A1R2 dipeptide as the most stable
conformational motif. Based on well-established biophysical principles
(Rajamani, Thiel, Vajda & Camacho, 2004), we defined this motif to be
the anchor of the interaction responsible for molecular recognition of
Cav2.2. Thus, we used the
A1R2 ensemble as scaffold to design
pharmacophore models to predict first-in-class compounds to disrupt
CRMP2-Cav2.2 interaction. Screening 27 million
commercially available compounds led to the identification of a
first-in-class peptidomimetic
((3R)‐3‐acetamido‐N‐[3‐(pyridin‐2‐ylamino)propyl]piperidine‐1‐carboxamide;
hereafter designated as CBD3063) that disrupted the
Cav2.2-CRMP2 interaction, inhibited
Cav2.2 function, and reversed experimental neuropathic
pain.