Lecture 14: Voltage Gated Ion Channels and Pain (2):

What Approaches Have Recently Been Used to Target Ca_v and Nav Channels in Pain Treatment?

• Small molecule drugs

• Disrupting channel trafficking

• drug repurposing

• Toxins

• Antibodies

How are small-molecule drugs used to target Cav channels for pain treatment?

• Clinical focus on the development of state-dependent blockers that selectively target ion channels in pathological states (pain) while preserving underlying nociceptive pathways to allow beneficial pain pathways to operate as warnings for injury and disease

• Mechanism: target Cav channel function and expression at the membrane to reduce Ca²⁺ influx and decrease neurotransmission, alleviating pain.

• Targets:

  • Cav2.2 (N-type): regulates sensory neuron neurotransmission.

    • C2230 → use- & state-dependent blocker, novel binding site.

    • RD2 → voltage- & state-dependent blocker, competes with ziconotide site.

  • Cav3.2 (T-type): coumarin-derived compounds, e.g., Toddaculin → T-type selective.

What is C2230, and how was it identified?

• An aryloxy-hydroxypropylamine

• A preferential use and state-dependent blocker of Cav2.2 channels → mitigates pain behaviours in a range of pain models

• Identified following the screening of >4200 compounds, assessing their ability to selectively bind Cav2.2.

  • Molecular docking analysis, in silico analysis, and site-directed mutagenesis were performed

• It induces use/frequency-dependent inhibition of Cav2.2 selectively

How does C2230 selectively inhibit Cav2.2 channels?

• Induce use/frequency-dependent inhibition

• It causes state-dependent block → block was enhanced at depolarised membrane potentials (- -50 mV) compared to normal.

  • preferentially targets inactivated channels at strongly depolarised Vm.

• Reduces Cav2.2 current → seen across a range of tested potentials → robust inhibiton seen

• No effect on voltage-dependent activation, but steady-state inactivation shifted leftwards, indicating fewer channels available to open in response to depolarisation in the presence of the SMD

  • shown using activation curves & normalised conductance against voltage plot

• This is achieved by the Cav2.2. channel is trapped and stabilised in a slow recovering state → takes longer to recover from the inactivated state (slow repriming channel)

• The drug accelerates the rate of open state inactivation, increasing the rate at which the channel inactivates and takes longer to recover

• It selectively inhibits Cav2.2 during high-frequency stimulation while sparing other voltage-gated channels → reduces Ca²⁺ influx selectively in sensory neurons.

What are the key effects of C2230 on Cav2.2 channels and pain behaviours in animal models

• CC2230 Inhibits Cav2.2 currents in DRG & TG neurons across rats, marmosets, humans (GPCR-independent).

• This reduces excitatory postsynaptic currents generated by the channel and neurotransmitter release in the spinal cord (inhibitory effect)

• In animal models, this:

  • Relieved neuropathic, orofacial, and osteoarthritic pain-like behaviours via 3 different administration routes.

  • Mitigates aversive responses to mechanical stimuli after neuropathic injury.

• It preserves protective pain responses (warning of injury/disease). without impacting motor or cardiovascular function in animals (reasonable safety profile)

How Does C2230 Bind to Cav2.2 Channels?

• Unlike typical Cav2.2 inhibitors, which bind in the pore region to residues within the pore lining and TMD III/IV fenestration, C2230 binds to key residues that point away from the pore and TMDIII/IV fenestration

• The identification of this novel binding sites allows for potential drug refinement, increasing affinity and subtype selectivity

What is RD2 and How Was it Identified?

• An orally available all-D-enantiomeric peptide that targets the pore region of Cav2.2

• Identified via a non-pain-related drug development program

• It competes with the zinconotide binding site at [nM]

  • At concs > 10 μM other subtypes (Cav1.2 and Cav3.2) are affected

• In a sciatic inflammatory neuritis model (In vivo proof of concept experiment)

  • Low oral dose (5mg/Kg): successful in alleviating NP pain

  • High oral dose (50mg/Kg) required to reduce pain in acute thermal response (tail flick test)

• Shows potential for further optimisatoin into a promising new drug candidate

How Does RD2 Inhibit Cav2.2.?

• It reversibly inhibits Cav2.2 in a concentration-dependently, voltage- & state-dependent manner

• It causes a reduction in current (Seen in the IV relationship)

• No effect on voltage-dependent activation → No change in threshold of activation (Act. curve shows leftward shift, but not statistically significant)

• Steady state inactivation is reduced → fewer channels available to open in response to depolarisation (Leftward shift in activation curve)

• Greater inhibition and blockade at depolarised potentials (-60 mV) than hyperpolarised (-100 mV) → voltage-dependent block

• Recovery is from inhibition and block is slower at depolarised potentials (-60 mV) than hyperpolarised (-100 mV) → state-dependent block

• This inhibits Ca²⁺ influx through Cav2.2, reducing excitatory neurotransmission in sensory pathways and pain transmission → reduced pain

What is an important caveat to consider when interpreting studies on Cav2.2 inhibitors?

• Many studies were done in heterologous systems expressing only the Cav2.2/β4 complex, without the α2δ subunit, so the results may not fully represent the behaviour of the complete native channel complex

• α2δ is crucial for channel trafficking and biophysical properties

  • No α2δ → impacts the functional properties of the channel and the ability of B subunits to modulate channel proteins

What are recent strategies for developing selective Cav3.2 inhibitors?

• Cav3.2 is widely expressed, making selective targeting difficult.

• Rosetta-based structural modelling using the Cav3.1 cryo-EM structure to identify compounds that would target the channel → identified coumarin-derived compound

  • Toddaculin (coumarin-derived): selectively inhibits Cav3.2, reduces DRG excitability, alleviates neuropathic, inflammatory & visceral pain in rodents.

    • Toddaculin refined and modifed to generate related compounds

  • Glycocoumarin (related compound) produced more potent inhibition and analgesic activity than the parent compound, shifted the dose-response curve right, and blocks Cav2.2 as well.

• Mechanism: Carbonyl oxygen on the drug interacts with Leu1508 in TMD III (within the VSD) of the pore,

  • novel drug interaction site for modulators and inhibitors → hotspot for drug design.

• Structural hotspot allows refinement of Cav3.2 inhibitors and pharmacological probes.

Why Have Many Promosing Small Molecule Drugs Failed At Clinical Trials?

• Despite showing promise in animal trials, candidates (e.g. Trox-1, Z160) fail at clinical trials due

  • failure to account for side effects in humans

  • Limited efficacy → reduced efficacy in certain types of pain

  • Poor pharmacological properties

• Thus, it is important to determine how newer drug candidates will overcome these problems, e.g. development of small-molecule drugs and targeting novel sites in the pore regions and VSD

• This is a probem for both Cav and Nav targeted drugs

How can ion channel trafficking be targeted in pain treatment?

• Disrupting trafficking and transcription of ion channels can be used to reduce the surface expression of ion channels → indirectly reducing excitatory channel activity

• Rationale: Chronification of pain caused by persistent stimulation of the nociceptive pathway due to an increased expression of excitatory channels; reducing surface expression can alleviate hyperexcitability.

• Various gene therapy approaches have been used to disrupt surface expression of channels/receptors involved in nociceptive signalling.

What Gene Therapy Approach Was Used to Selectively Target and Reduce The Surface Density of HVA Cav Channels DRG Neurons?

• A Ca_v-aβlator targeting the ubiquitination of HVA Cav channels was used to reduce the development of neuropathic pain

• This Ca_v-aβlator construct consisted of a nanobody (nb.F3) targeted to the Ca_vβ subunit fused to the catalytic HECT domain of Nedd4-2 E3 ubiquitin ligase (nb.F3-Nedd4-2)

  • CavB is only associated with HVA channels, not LVA (selective targeting)

• When expressed in cells, the nanobody attached to the beta attaches to the pore-forming subunit of Cav2.2 at the cell surface

• Ned-2 promotes the ubiquitination of the whole channel complex, resulting in the endocytosis of the Cav2.2 channel, reducing its surface expression

• This allows for the selective targeting of HVA Cav channels for ubiquitin-mediated degradation, reducing the specific cell surface channel rather than the total protein level of the channel

What Was the Effect of the Ca_v-aβlator Gene Therapy Approach In a Mouse?

• Subcutaneous injections of adeno-associated virus encoding Cav-aβlator construct into the hind paw of mice resulted in

  • Robust inhibiton of HVA currents

  • Increased frequency of IPSC (Inhibitory post synaptic currents) in the dorsal horn

  • Long-lasting relief in NI-induced pain without apparent adverse effects to the animal

Why is the Ca_v-aβlator Gene Therapy Approach a Good Strategy for Targeting Cav Channels?

• Targets the Beta subunit and so the Cav channel using a nanobody with ubiquitin ligase to target the channel for ubiquitination, using the cell machinery to reduce surface expression.

• Potential application for pre-emptive Analgesia, to prevent post-operative pain by reducing Cav channel activity in specific areas.

• More effective than SMDs targeting α1/β interactions, which need high doses for competitive inhibition.

• The construct is genetically encoded, allowing for directed and targeted expression in specific cell populations, reducing off-target effects.

• This restricted expression is mediated using specific promoters in the AAV construct, which allows targeting of high-voltage activated (HVA) neurons, particularly in the DRG, minimising side effects in other cells.

• This work has yet to extend into clinical settings but shows promise for using GT to selectively deliver therapeutic constructs.

What is CBD3063?

• A CRMP2-derived small molecule peptidomimetic of Cav2.2. N-type Ca channels

• Derived from the CRMP2 Ca channel binding sequence → disrupts binding between CRMP2 and the ion channel

  • Normal: CRMP binds to an ion channel, e.g. Cav2.2.; SUMOylation of CRMP2 increases channel expression at the cell surface

• Compounds ats to disrupt CRMP2 binding to Cav2.2, reducing channel surface density/number of channels at the surface

• This intern decreases Ca2+ influx, NT release and pain through the promotion of endocytosis

• A first-in-class small molecule, allosterically regulating Cav2.2, by disrupting CRMP2 interaction for analgesia and pain relief w/o negative side-effect profiles in animal models

  • Potentially more effective alternative to GBPs

What is the Structure of CBD3063?

• A 15-amino acid peptide (CBD3 → Ca channel Binding Domain 3) was identified from studying CRMP2 binding to Cav2.2.

  • CBD3 peptide interacts specifically with Cav2.2 channels.

• The peptide contains 2 important residues: alanine (A) and arginine (R).

  • The A1R2 dipeptide (alanine + arginine) is essential for Cav2.2 binding

• The first 6 amino acids of CBD3 have an anti-nociceptive (pain-reducing) core.

  • A1R2 dipeptide is important in regulating Cav2.2 binding affinity.

How Was CBD3063 Identified?

• Used A1R2 dipeptide to design pharmacophore models for screening compounds.

  • Screened 27 million compounds for activity against the A1R2 anchoring motif.

  • Found 200 hits, with 77 compounds tested based on depolarisation-evoked Ca²⁺ influx in DRG neurons.

  • 9 small molecules tested by electrophysiology.

  • CBD3063 was evaluated both biochemically and behaviorally.

What is the Effect of CBD3063 in Animal Models (Rat and Mice)?

• Reduced response to mechanical stimulation

• Reversed Neuropathic Pain and Inflammatory Pain across sexes

• No changes in sensory, sedative, depressive or cognitive behaviours

  • Compared to opioids and gabapentinoids, this drug could be more effective and have fewer side effects

How Could Post-Translational Modifications Be Used to Reduce Cav3.2 Surface Expression?

• Idea: Channel trafficking, surface expression and function are modulated by at least 3 key post-translational modifications (N-linked glycosylation, phosphorylation, ubiquitination)

• Strategy: Manipulation of these post translational modifications is successful in reducing Cav3.2 currents and surface expression, reducing chronic pain in animal models (mechanical allodynia, diabetic peripheral neuropathy)

What are the 3 Main Post-Translational Modifications, and What is the Effect on Ca3.x Channels When Manipulated?

• Glycosylation: Addition of sugar groups to asparagine residues on Cav3.x promotes proper folding, increasing surface expression.

  • Blocking glycosylation reduces current and pain in DPN models (no effect in normal mice).

• Phosphorylation of Cav3.2 is regulated by kinases like CaMKII, PKA, PKC, and Cdk5.

  • Cdk5 increases Cav3.2 surface expression after nerve injury.

  • Inhibiting Cdk5 (e.g., olomoucine) reduces Cav3.2 current and reverses mechanical allodynia in rodent models.

• Ubiquitination targets Cav3.2 channels for degradation via proteasomes.

  • USP5 (deubiquitinase) increases in inflammatory and NP pain models;

  • Blocking USP5 or its interaction with Cav3.2 reduces Cav3.2 surface expression in 1° afferents in the dorsal horn → reverses allodynia in chronic pain models (e.g., neuropathic pain).

What Approaches Have Been Used to Target Nav Channels as a Treatment for Pain

• 1st Generation Non-selective channel blockers, e.g. local anaesthetics

• Drug repurposing, e.g. carbamazepine

• 2nd generation subtype selective blockers

• Biologics

  • Antiboies

  • Toxins

  • Antibody-Toxin conjugates

What are Local Anaesthetics?

• First-generation non-selective Nav blockers (e.g. lidocaine, phenytoin)

  • Pan-selective → targeting any available Nav channel

• Generate a non-selective block of Nav channels in Aδ and C-fibre nociceptive neurons

• Cause a loss of sensation due to pan-Nav inhibition → reduce AP firing → useful in reducing pain

How Do Local Anaesthetics Inhibit Nav Channels?

• Produce use-/frequency-dependent block

  • Most effective during high-frequency firing

• Bind to the gating regions (S6 segments) of TMDs I, III & IV

• Push and stabilise channels in the inactivated state

  • Ideal for targeting channels during periods of high activity (pathological) pain signalling

• Low doses used systemically are effective in pain release

  • High doses → toxic and death due to non-selective Nav blockade

How is carbamazepine used in pain treatment?

• Originally developed as an anti-epileptic

• Targets Nav channels via voltage- and use-dependent block

• Binds to the local anaesthetic (LA) binding site with high affinity

• Current FDA-approved first-line treatment for trigeminal neuralgia → drug repurposing

• Also used in pain channelopathies (e.g. IEM)

How does carbamazepine act on mutant Nav channels in inherited erythromelalgia (IEM)?

• Carbamazepine has a different mechanism of action depending on the mutation present

• In certain Nav1.7 and Nav1.8 gain-of-function mutants (S241T, V400M, I234T), carbamazepine does not act via use-dependent block

• Instead, it acts as an activation modulator (of specific IEM mutant Nav1.7 and Nav1.8 channels)

• Causes rightward shift in the activation curve → larger depolarisation required to open the channel

  • In the presence of the drug, it becomes harder to open the channel

• Counteracts the pathological hyperexcitability of increased channel opening and increases Na⁺ influx caused by IEM mutations

• Acts to functionally restore mutant channels toward WT Nav channel behaviour

What are 2nd-generation Nav channel blockers, and why were they developed?

• Developed to overcome off-target effects generated from the broad-spectrum action of 1st-generation non-selective Nav blockers

• Aim to be subtype-selective, rather than pan-selective Nav inhibitors

• Focus on Nav1.7 as a key target:

  • Loss-of-function mutations → congenital insensitivity to pain (CIP)

  • Suggests selective Nav1.7 inhibition could provide analgesia

• Extensive pre-clinical evidence for small-molecule with blocking activity against Nav1.7/Nav1.8

  • Limitation: as many candidates lack true subtype selectivity in practice

What is PF-05089771?

• A 2nd generation Nav1.7 subtype selective blocker → an arylsulfonamide that targets and modulates the VSD in TMD IV

  • It exhibits 1000-fold selectivity for Nav1.7 vs other subtypes

• Preliminary evidence of reduced pain in IEM patients

• Small-scale study: Low efficacy in diabetic peripheral neuropathy and dental pain

  • Highlights the need to test drugs on different types of pain

Why did PF-05089771 fail to show efficacy in broader pain indications (Dental Pain and Diabetic Peripheral Neuropathy) ?

• Poor CNS penetrance → access to the central terminals of nociceptors

• Suboptimal Pharmacokinetic/Pharmacodynamic properties

• Issues with bioavailability

  • The compound failed and did not pass phase II Clinical Trials

• Highlight the need to:

  • Test drugs across multiple pain states

  • Consider central vs the peripheral site of action

What is Vixotrigine (CNV1014802 / BIIB074) and Why Did it Fail At clinical trials?

• Voltage-gated Na⁺ channel blocker developed for neuropathic pain, particularly trigeminal neuralgia

• Predominantly targets Nav1.7, but also blocks Nav1.6, Nav1.2, and Nav1.8 → poor subtype selectivity

  • Nav1.8 contributes to AP upstroke and pain transmission

  • Nav1.2 is not important in pain pathways

• Showed promising analgesic effects in small-scale clinical trials

• Advanced to Phase III, but failed larger clinical trials (2018)

• Highlights the challenge that insufficient Nav subtype selectivity limits clinical efficacy

Why have large-scale clinical trials of Nav1.7 blockers failed to show consistent analgesia and caused autonomic side effects?

• Variable drug properties: Differences in bioavailability, therapeutic window, and pharmacodynamics

• Heterogeneity in pain assessments: Different studies assess different types of pain and have different pain endpoints

• Subtype and tissue distribution

  • Nav1.7: DRG, TG, SG, CNS, PNS, sympathetic ganglia

  • Nav1.8: mainly DRG & TG
    → Blocking Nav1.7 affects non-pain pathways

• Autonomic side effects due to the broad distribution of Nav1.7 in CNS neurons and PNS/SNS ganglia → sympathetic and central side effects

• This demonstrated the need for subtype-selective drugs and drove interest into biologics (antibodies, nanobodies, peptides, peptidomimetics)

What are the advantages and limitations of biologics (antibodies/peptides) compared with small-molecule drugs?

• Advantages:

  • Higher binding selectivity → approaching true specificity

  • Abs and peptides are metabolised via normal protein dynamics and pathways

  • Avoid drug–drug interactions and metabolism-related toxicity

• Limitations:

  • Poor membrane permeability

  • Poor CNS penetration

  • Risk of adverse immune reactions

What are the success rates of biologics versus small molecules, and how is biologic targeting of ion channels demonstrated in nature?·   

• Biologics have a higher success rate (13.2%) compared to small molecules (7.6%) from Phase I to FDA approval (2019 data).

• Ion channel targeting with biologics has been successfully demonstrated in nature (e.g., venoms from cone snails, spiders, scorpions, sea anemones).

• Example: Ziconotide, a Cav2.2 blocker, is used clinically as a selective treatment for chronic pain, proving the concept of biologics for pain management.

How Can Toxins Target Nav Channels?

• Toxins can be naturally occurring peptides and small molecules

• Depending on where the peptide/ toxin targets it can act as

  • pore blockers (e.g., TTX)

  • gating modulators (inhibitors/activators, e.g., μ-conotoxins).

• Many toxins, e.g. spider toxins, are subtype-selective, targeting specific Nav channels e.g. Protoxin 11 selectively inhibits Nav1.7, useful for pain research.

What is the status of Nav-targeted toxins as a treatment for chronic pain?

• Toxins tested for their use in chronic pain treatment pre-clinically and in clinical trials

  • Peptides such as TTX and Protoxin 11 target Nav1.7 and Nav1.8.

• Looked to targets a range of ion channels involved in inflammatory neuropathic pain, nerve injury–induced NP, and diabetic neuropathy.

• None have reached clinical use.

What advantages do antibodies have over peptides when targeting ion channels?

• Desirable pharmacokinetics due to immunoglobulin structure.

• Can induce pore block, allosteric modulation, or gating effects to reduce Na⁺ influx.

• Can be enginnered

  • Antibody-dependent cytotoxicity.

  • Conjugation with toxic or radioactive “warheads” to selectively target and reduce channel function

  • Peptide–antibody conjugates combine the useful properties of selective channel targeting (from peptide) with efficient delivery (via Ab).

How can antibodies modulate ion channel function?

• Depending on the epitope targeted, antibodies can mediate:

  • Internalisation of ion channel–antibody complexes.

  • Modulation of channel function via conjugation with peptides or small-molecule toxins.

• They can act through one or a combination of these mechanisms.

What is the Nav1.7 toxin–antibody conjugate (GpTx-mAb) and how was it optimised?

• GpTx-mAb combines an anti-2,4-dinitrophenol human IgG1 antibody (carrier) with the peptide toxin “warhead” GpTx-1 (tarantula peptide toxin).

• Site of Ab toxin conjugation was determined by assessing the reactivity of 13 potential cysteine conjugation sites on the antibody to determine the best in potency

• Variations in peptide attachment site, linker length, and peptide loading conditions were used to find the most potent combination.

• Aimed to prolong channel block and reduce the frequency of dosing.

What are the key effects and advantages of the Nav1.7 toxin–antibody conjugate in vivo?

• In vivo half-life extended by 130-fold vs “naked” peptide → conjugation with Ab allows prolonged channel block

  • Clinical implications: potential for reduced dosing frequency; important where peptides are selective but short-lived and rapidly broken down by proteases

• Better distribution to mouse DRG & sciatic nerves compared with parent mAb.

  • Peptide highly selective for Nav1.7 → better delivery to target (Nav1.7 DRG neruons)

• Significant Nav1.7 block of WC current, nearly as effective as TTX.

• i.v. Administration demonstrates clinical potential for selective Nav1.7 inhibition.

• combination of pharmacokinetic benefits from antibodies with the selective potency of peptide toxins.

How can antibody-toxin conjugates improve targeting of ion channels?

• Use of

  • an antibody for favorable pharmacokinetics and delivery.

  • Selective peptide toxin (“warhead”) for potent channel inhibition properties.

• Optimisable via conjugation site, linker length, and peptide loading.

• Potential for longer half-life, improved tissue targeting, and reduced dosing frequency.