Discussion
Structure-based drug design
allows the optimization of a chemical structure in which the structure
of a protein is used as the basis to identify or design new chemical
compounds that are predicted to bind to a target (Aparoy, Reddy &
Reddanna, 2012). Alternatively, ligand-based approach is used in the
absence of the protein structure and relies on knowledge of molecules
that bind to the biological target (Aparoy, Reddy & Reddanna, 2012). To
disrupt the Cav2.2–CRMP2 interaction, we had neither a
structure of the complex nor any drug target data. Thus, based solely on
the sequence of the CBD3 peptide, we resorted to first principles to
identify the structural motif that triggers this interaction. Recently,
we tackled a similar problem whereby using MDS, we identified the
residue anchoring the interaction between a peptide derived from the
disordered N-terminal of Kv2.1 and syntaxin1a, which in
combination with syntaxin crystal structure led us to the discovery of a
first-in-class small molecule neuroprotectant (Yeh et al., 2019).
In this study, we developed a novel pipeline that leverages the
computational power of MDS to identify the most stable conformational
motif of a peptide, i.e., CBD3, and then used it to develop a
pharmacophore model to generate peptidomimetics – a medicinal chemistry
approach where parts of the peptide are successively replaced by
non-peptide moieties until a non-peptide small molecule is discovered
(Perez, 2018). Peptides present desirable medicinal properties like
predictable metabolism, good efficacy, safety, and tolerability;
however, they are chemically and physically unstable due to rapid
proteolysis and inadequate membrane permeability (Fosgerau & Hoffmann,
2015). Notably, editing peptide sequences to develop peptidomimetic
analogs creates a promising class of therapeutics that can have inherent
advantages, including oral administration, good membrane penetration
ability, and enhanced biological activity (Smith, 2015). Considering
these strengths, our model predicted first-in-class compounds to disrupt
the Cav2.2–CRMP2 interaction. Specifically, we screened
more than 27 million commercially available compounds in the ZINC
database using the open access server ZincPharmer and identified 77
suitable compounds for experimental testing. The primary in vitroscreening identified 9 compounds that inhibit Ca+2influx by more than 50% relative to control. Furthermore, analysis of
the shared pharmacophores among the 9 compounds permitted us to predict
active chemotypes that when screened against MDS of CRMP2-derived
peptides allowed us to fully rationalize active from inactive peptides.
The latter provides a strong rationale for our method and its potential
application for other targets. From a biological point of view, we show
that by disrupting the Cav2.2–CRMP2 interaction,
CBD3063: (i) inhibits Ca2+ influx in rat DRG neurons,
(ii) decreases the functional activity of N-type Ca2+channels, (iii) reduces membrane expression of Cav2.2,
(iv) does not affect the activity of other voltage-gated ion channels,
(v) reduces sensory neuron excitability, and (vi) is antinociceptive in
rats with a spared nerve injury model of pain.
Cav2.2 channels are almost expressed exclusively in
neuronal tissue (Nowycky, Fox & Tsien, 1985) and are abundant at
presynaptic nerve terminals where they trigger the release of
neurotransmitters such as glutamate, calcitonin gene-related peptide,
and substance P (Evans, Nicol & Vasko, 1996) via physically interacting
with the synaptic release machinery (Zamponi, 2003). For this reason,
Cav2.2 channels are critical determinants of increased
neuronal excitability (Yang et al., 2018) and neurotransmission that
accompany chronic neuropathic pain (Cizkova et al., 2002). In 2018, the
Dolphin laboratory (Nieto-Rostro, Ramgoolam, Pratt, Kulik & Dolphin,
2018) reported a mouse expressing Cav2.2 channels with
an extracellularly accessible hemagglutinin epitope tag engineered into
their pore forming Cav2.2 α1 subunit
(Cav2.2_HA) permitting, for the first time,
identification of endogenous Cav2.2 channels in the
plasma membrane of sensory neurons. These mice revealed
disease-associated changes in the subcellular distribution of
Cav2.2 in the pain pathway that confirmed the importance
of these channels as suitable targets for development of novel pain
therapies. Consistent with these findings, the therapeutic potential of
targeting Cav2.2 has been demonstrated in
Cav2.2 deficient mice which have reduced responses to
mechanical stimuli, radiant heat, and chemical-induced inflammatory pain
(Hatakeyama et al., 2001; Kim et al., 2001) and in nociceptive neurons
specifically expressing the exon 37a variant of Cav2.2
(Cav2.2e[37a]) mice that display increased N-type
Ca2+ currents and open channel probability when
compared to neurons that only express the exon 37b variant of
(Cav2.2e[37b]) (Bell, Thaler, Castiglioni, Helton &
Lipscombe, 2004). In these mice, in vivo silencing of
Cav2.2e[37a] prevented the development of mechanical
allodynia and thermal hyperalgesia, demonstrating that targeting
Cav2.2e[37a] channels by using splice
isoform-specific gene silencing is an effective means for controlling
the transmission of thermal and mechanical stimuli in pain conditions.
Despite compelling genetic evidence of the importance of
Cav2.2 in pain, clinical development of N-type
Ca2+ channel blockers have proven to be challenging.
Although modulators of Cav2.2–α2δ interaction (i.e.,
Gabapentin and Pregabalin) are recommended as first-line treatment for
neuropathic pain (Attal et al., 2006), they only partially alleviate
chronic pain, are implicated in overdose deaths (Kuehn, 2022), and cause
a litany of side effects (Zamponi, Striessnig, Koschak & Dolphin,
2015). A Cav2.2-selective, state-dependent inhibitor –
N-triazole oxindole (TROX-1) – was reported by Merck but cardiovascular
and motor impairment hampered its further development (Abbadie et al.,
2010). Another study reported a sulfonamide-derived, state-dependent
inhibitor of Cav2.2, but this compound was limited by
structural liabilities of this class of compounds (Shao et al., 2012).
Targeting other interacting partners of Cav2.2 can also
result in reversal of pain symptoms. For example, the β3 auxiliary
subunits interact with Cav2.2 channels (Ludwig,
Flockerzi & Hofmann, 1997; Scott et al., 1996) to speed up their
activation, increase their membrane localization, and increase
neurotransmitter release (Richards, Butcher & Dolphin, 2004; Welling et
al., 1993). Expression of β3 protein increases in neuropathic pain (Li
et al., 2012) and β3-null mice exhibit suppressed pain responses due to
decreased Cav2.2 currents (Murakami et al., 2002).
Consistent with these findings, we reported that targeting the
Cav2.2–β interaction reduces currents through
Cav2.2 channels, inhibits spinal neurotransmission, and
alleviates neuropathic pain (Khanna et al., 2019). Collectively, these
studies converge on the idea that targeting auxiliary subunits of
Cav2.2 channels is beneficial for pain management. Along
these lines, accumulating evidence points to CRMP2 as a new auxiliary
subunit of Cav2.2 channels (Striessnig, 2018).
Over the past decade, we have shown that interrupting the interaction
between Cav2.2 and CRMP2 with CBD3 peptides is
efficacious in reversing pain. We reported that disrupting
Cav2.2 –CRMP2 binding with tat-CBD3 or myr-tat-CBD3
peptides does not affect memory, motor functions, or anxiety/depression,
and does not produce any addictive behaviors (Brittain et al., 2011b;
Francois-Moutal et al., 2015). Likewise, interrupting this interaction
has neuroprotective effects (Brittain et al., 2011a; Brustovetsky,
Pellman, Yang, Khanna & Brustovetsky, 2014; Ji et al., 2019). Tat-CBD3
(Brittain et al., 2011a; Brustovetsky, Pellman, Yang, Khanna &
Brustovetsky, 2014) and R9-CBD3 (Ji et al., 2019) also disrupts the
CRMP2–NMDAR interaction. As a result of this, both peptides attenuate
NMDAR-mediated currents, have neuroprotective effects against
glutamate-induced Ca2+ dysregulation (Brittain et al.,
2011a; Brustovetsky, Pellman, Yang, Khanna & Brustovetsky, 2014) and
protect against neurotoxicity caused the toxic fragment of amyloid-β
(Aβ)25-35 (Ji et al., 2019). These studies demonstrate
that interfering with Cav2.2-CRMP2 binding is safe,
beneficial, and does not produce unwanted side effects.
We previously reported that a single amino acid point mutant of CBD3
(tat-CBD3A6K) decreased R- and T-type
Ca2+ currents in DRG neurons (Piekarz et al., 2012),
and that tat-CBD3 peptide also decreased T-type currents (Piekarz et
al., 2012) in sensory neurons. Importantly, in the present study,
CBD3063 did not exert an effect on any other voltage-gated calcium
channel, which argues for selectivity of our peptidomimetic compound for
Cav2.2. This is in line with our previous observations
that the activity of low-voltage-activated Ca2+channels is independent of CRMP2 (Cai, Shan, Zhang, Moutal & Khanna,
2019). We have shown that Cdk5-mediated CRMP2 phosphorylation at residue
S522 increases its binding to Cav2.2, which leads to an
increase in calcium influx (Brittain, Wang, Eruvwetere & Khanna, 2012).
To discard a potential effect of CBD3063 on CRMP2 phosphorylation, we
measured pS522 CRMP2 and found that CBD3063 did not affect this
posttranslational modification. This data correlates with our previous
findings that interrupting CaV2.2-CRMP2 interaction with myr-tat-CBD3
does not affect CRMP2 phosphorylation (Francois-Moutal et al., 2015).
Along the same lines, we investigated the regulation of sodium channels
by CBD3063 since we previously described that phosphorylation and
SUMOylation of CRMP2 regulatesNav1.7channel trafficking and activity (Dustrude, Moutal, Yang, Wang, Khanna
& Khanna, 2016; Dustrude, Perez-Miller, Francois-Moutal, Moutal, Khanna
& Khanna, 2017; Dustrude, Wilson, Ju, Xiao & Khanna, 2013). In the
present study, we found that CBD3063 does not directly affect currents
through sodium channels, or indirectly through an effect on CRMP2
phosphorylation or CRMP2 SUMOylation (data not shown), indicating that
this regulation is unaffected by CBD3063 treatment. These data, together
with the lack of effect on K+ currents, suggest that
targeting this interaction with CBD3063 does not result in inhibition of
other ion channels relevant for pain signaling or in dysregulation of
CRMP2 posttranslational modifications.
Despite the documented success of CBD3 peptide in achieving analgesia
without side-effects, peptides are nevertheless hampered by (i) short
half-life caused by their poor in vivo stability (Bruckdorfer,
Marder & Albericio, 2004) which may be attributed to the presence of
numerous peptidases and excretory mechanisms that inactivate and clear
peptides, and (ii) negligible bioavailability caused by digestive
enzymes that are designed to break down amide bonds of proteins and
cleave the same bonds in these peptides. To circumvent some of these
problems, in the present work we utilized our pharmacophore modelling to
generate small molecule peptidomimetics to improve upon the biological
activity of the CBD3 peptide by mimicking the chemical features
responsible for bioactivity with enhanced drug-like properties. As a
result, in contrast to the short-lived actions of CBD3 peptides, the
peptidomimetic developed here (i.e., CBD3063) exhibited prolonged
antinociceptive profile following a single intrathecal administration as
well as long-lasting (>14 days) reversal of mechanical
allodynia with repeated intrathecal dosage in rats with chronic
neuropathic pain. The latter also points to a lack of tolerance to
CBD3063. The prolonged antinociceptive effect of CBD3063 could be
attributed to the significant decrease of action potential firing in
sensory neurons that could potentially translate into a decrease in
spinal cord excitability and neurotransmitter release (not tested here).
These findings are congruent with those observed with an adenoviral form
of a mutant CBD3 (i.e., AAV6-CBD3A6K), which reduced the
firing of dorsal horn neurons and reversed mechanical allodynia and
thermal hyperalgesia for up to 6 weeks following intraganglionic
delivery of the virus in rats with tibial nerve injury (Yu et al.,
2019a).
Here, we have presented a translational workflow that uses structural
modeling to direct the resolution of protein-protein interactions
involving poorly characterized disordered domains, resulting in both
mechanistic insights and the identification of an analgesic. This
translational workflow led to the discovery of CBD3063 as a
first-in-class, CRMP2-based peptidomimetic, which selectively regulates
Cav2.2 to achieve analgesia. We note that although CBD3063 exhibits
promising drug-likeness (QED score, Table 1 ) it does have
marginal predicted blood-brain barrier penetration (BBB score,Table 1 ). To improve to CNS exposure, CBD3063 can be further
optimized to lower the topological polar surface area by, for example,
modifying and/or removing the acetamide and urea bonds. Another option
to increase hydrophobicity is to introduce aromatic group (e.g., in the
acetamide area) or to strategically position fluorine atoms to lower
capacity of N-H donor (e.g., as 3-fluro-2-aminopyridyl moiety). Overall,
we intend to explore targeted changes in CBD3063 to improve its
predicted ADME properties while maintaining its current biochemical
profile.