Lecture 16: Selective Neurotoxin

Why Have Animals Developed Venom Systems?

• To defend (protection)

• To deter

• To capture prey

• Animal toxins can target all aspects of physiological pathways (from a single venom), including

  • Neurotoxins – Target neurons

  • Target ion channels to enhance or inhibit activity

  • K⁺; Na⁺; Ca²⁺; ligand gated nAChR; NMDA receptor; ASICs

What is Venomics?

• Individual toxins within venoms are isolated and analysed

• A single venom can contain 40-50 different components, which allow the venom to target particular areas

  • Typically have mixed effects to disrupt a range of physiological processes

What is the Site of Action of Neurotoxins and How Has This Been Exploited?

• Have activity at a range of sites across the body, e.g. ion channels and receptors

• Components of venoms have been used as scientific tools to investigate ion channels for their

  • stoichiometry,

  • binding sites

  • gating characteristics (following point mutations)

• Also, allow for the precipitation and isolation of ion channels for the identification of different ion channel subfamilies

  • Helped in understanding the activities of Na, K and Ca channels

What is the Structure of A Toxin?

• Particular structure

• Small peptides, differing in length → normally 8-70aa long

• Compact small scaffold structure → exists as alpha and beta sheets

  • Gives rise to a 3D conformation → compact structure

• Highly compact structure stabilised by disulfide bridge and hydrogen bonds

  • ~2-4 disulfide bridges present

• High specificity, high activity (potency), but no accumulation in the body

How have venoms contributed to drug development?

• Venoms contain multiple toxins with different biological actions

• These diverse effects provide potential targets for therapeutic development

• Example: Captopril (an ACE inhibitor) was developed from snake venom components

Why are Snake Venoms Currenlty of High Research Interest?

• ~40 + peptides isolated from the venom of black and green mamba snakes

• Have lots of toxins present within the venom, which can attack particular ion channels in the:

  • Heart - Cardiotoxins

  • Muscle -Myotoxins

  • Enzymes (phospholipase A2-like, proteases)

  • Neurons – Neurotoxins

    • a-neurotoxins

What Neurotoxins are Unique to Mambas?

• Fasciculins, dendrotoxins (bind Ca2+ channels), two calcium channel blockers, and muscarinic toxins

• Mambalgins: bind to acid-sensing ion channels and produce pain relief

What are the Acid Sensing Ion Channels?

• Ion channels are widely expressed throughout the body

• Usually activated by extracellular acidic pH (protons)

  • Increase in H+ observed in infection, cell trauma (events that initiate pain response)

• Mediate a Na+ selective current

• Ion channel family: ASIC1-4

• Inhibited by neurotoxin Mambalgins

What are the different families of ASIC Channels?

• Ion channel family: ASICs1-4

• There are different subunit expressions that make up the channels, determining central or peripheral localisations

  • ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 expressed in sensory neurones in the peripheral nervous system;

  • ASIC1a, ASIC2a, ASIC2b, ASIC4 are widely expressed in the central nervous system

• They can exist as homotrimers or heterotrimers of ion channels (depending on subunit composition)

What is the Structre of ASIC Channels?

• Size and binding sites of the channel are crucial determinants of its structure and selectivity

• ASIC extracellular portion contributes the greatest volume of the channel

• Channel is predominatly Na+ selective, but allows other postive ions through

• Following proton binding to the extracellular domain, the pore opens allowing ion passage

• This leads to excitations and activation of the pain pathway

What are the Main Types of Mambalgins?

• There are 3 different types

• They are similar in structure, differing by one amino acid at a particular point

• Have similar binding properties to particular ASICS

• Have 4 different disulfide bridges, giving rise to a distinct three-finger toxin conformation

• Belong to the three-finger toxin family

What is the Three Finger Toxin Family?

• Toxins with a distinct three finger conformation

• They have different properties and activities depending on the three-finger toxin

• Multiple members of this family, with different targets, including ASICs

• Mambalgins represent a new subfamily

  • Less than 50% homology to other 3FTXs

How Do Mambalgins Differ From Other Three Finger Toxins?

• They represent a new sub-family due to their distinct 3-finger conformation

• 3 beta-stranded loops, which can be differentiated from other TFTs

  • Mambalgins: Long Loop II (shorter Loop I, III)

  • Other TFTs: 3 beta-stranded loops of the same length

What is the Mechanism of Inhibition of Mambalgins?

• Inhibit ASICs activity by binding to a particular site in the acid sensing of the ion channel

• The acid-sensing area is located extracellularly, where the mambalgins bind, with their 3-finger structure facilitating the binding of toxins to the acid-sensing pockets

• This leads to a change in conformation, which stops pore opening and ion passage

How Do Mambalgins Inhibit ASICs?

• Depending on the ion channel subunit and composition, the toxins will have different affinities and IC50

• It reversibly inhibits:

  • rat ASIC1a (IC₅₀=21-55 nM),

  • rat ASIC1b (IC₅₀=103-192 nM),

  • rat ASIC1a-ASIC1b (IC₅₀=72 nM),

  • rat ASIC1a-ASIC2a (IC₅₀=246-252nM)

  • rat ASIC1a-ASIC2b (IC₅₀=61 nM)

    • slightly IC50s seen, but they are highly potent and effective at inhibiting the current

Why are Mabalgins Thought to be good for Pain Treatments?

• ASICs involved in different types of pain

• Blockage of ion channels by mambalgins can prevent pain signal transmission

• Positions toxins well for drug development, as current pain treatments for chronic pain are poor

• Opioids (current go-to treatment) are limited in effectiveness over time due to their side effects and abuse liability

What do acute inflammatory pain model graphs show about mambalgin vs morphine analgesia?

• Measure: time before paw withdrawal following thermal algesia (pain tolerance to heat)

• Vehicle ± Naloxone: low pain tolerance

• Morphine: longer period of contact with heat source→ strong and effective analgesia

  • Opioid-dependent → blocked by naloxone

• Mambalgin-1: effective but less potent than morphine

  • Not blocked by Naloxone → non-opioid mechanism

• Removal of ASIC1a channel → loss of mambalgin effect

• Analgesia reduced with PcTx toxin → confirms ASIC-dependent, non-opioid pathway

How does the route of administration affect the analgesic action of mambalgin-1?

• Intrathecal (CNS) administration produces central (supraspinal) analgesia

  • Required because the toxin does not cross the blood–brain barrier (BBB) → must be injected into CNS

• Intraplantar (paw) administration produces a peripheral analgesic effect

• Blocking ASIC1a (e.g. with PcTx) → reduces analgesia to vehicle level

• Peripheral analgesia is likely mediated via the ASIC1b subunit (different from the central mechanism)

How Does Mambalgins Analgesic Effect Compare to Morphine?

• Over time, morphine’s effect decreases, suggesting tolerance

• This tolerant effect is less pronounced with mambalgins (more prolonged and better analgesic effect observed over time)

• Opioids, e.g. morphine, cause respiratory depression → risk increases with continued use or overdose, whether given intrathecally or intraplantar

• This respiratory depression is not seen with mambalgin 1, even when given intrathecally

• Overall: mambalgins are better than opioids: produce good, sustained pain relief without respiratory depression

What are the Consequences of Mambalgins-Mediated Inhibition?

• Central inhibition of ASICs1a and ASICs2 heteromeric channels mediates analgesic effect

• Peripheral inhibition of ASIC1b (more predominant subunit in the periphery) and ASIC3 mediate the analgesic effect

• Multiple nociceptive stimuli can be blocked, including neuropathic pain (to which opioids have had limited success)

• Little evidence of tolerance

What Toxins Target Kv Channels?

• Dendrotoxins

• Margatoxin

• KAaH1 and 2 toxins

What are Dendrotoxins?

• Derived from the black mamba snake → one of the most specific and selective toxins

• Selectively block Kv1.1. channels (via alpha subunit)

• Pharmacological blockade of Kv channels leads to increased synaptic activity due to their role in excitable cells

• This leads to

  • Action potential broadening

  • Slowing of membrane repolarisation

  • Increased NT release

  • Increased excitability

• At high doses: seziures and death

What are the potential therapeutic applications of dendrotoxins?

• At low doses, it may have beneficial effects

  • Investigated for enhancing cognition via increased neurotransmitter release

• Target ion channels that are overexpressed in:

  • Cancer cells

  • Immune cells

  • Alveolar epithelial cells

• Mechanism: Blocking ion channels can restore normal physiological activity

What are Voltage Gated K+ (Kv) Channels?

• Largest and most complex family of ion channels

• There are Kv1-12 subfamilies

• Kv1.1 and Kv1.3 are the target of many toxins

Why are Kv1.1 and Kv1.3 Of Interest When Looking at Toxins>

• Many toxins are selective for these channels

• These channels can be expressed together, but this can change depending on the cell type

• These channels play a role in controlling membrane excitability, cell volume regulation, and modulation of intracellular K+ concentration

• They also impact on cell cycle progression, proliferation and apoptosis

What are Kv1.1 and Kv1.3 Channels Important In?

• Setting the membrane potential of the cell

• Volume regulation

  • These two functions are critical in G1/S phase → membrane excitability and extent of hyperpolarisation prepare the cell for the growth phase

  • They also regulate cell volume via K+ efflux → allows for shrinkage, which is important in preparation for cell growth

• Regulation of Ca2+ oscillations

• Participation in signalling complexes

How are Kv1.1 Cells Implicated in Cancer and How May Dendrotoxin Help Here?

• Overexpressed in some types of cancer cells

• Use of Dendrotoxin found to disrupt cell growth (anti-proliferative) in tumour cell lines and mouse tumours, only when injected into tumours (unable to cross barriers)

• Dendrotoxin K arrests the cell cycle by disrupting the G1-S transition phase

  • Selectively increases protein expression of cyclin-dependent kinase inhibitors p27^Kip1, p15^INK4B and p21^Waf1/Cip1

  • Decreases protein levels of cyclin D3

How Does Dendrotoxin K Affect Tumour Growth?

• It causes a suppression of tumour cell growth

• Interferes with membrane excitability, arresting the cell in G1/S phase and interfering with signalling pathways

• Visible differences in growth observed in tumours following dendrotoxin administration in tumours → cause a reduction in growth

What Evidence Supports the Use of Dendrotoxins In Cancer, Where Kv1.1. Cells are Implicated

• 10nm dendrotoxin reduces cell proliferation of MCF-7 breast cancer cell lines by 30%

• 100nm dendrotoxin reduces cell proliferation in chemoresistant non-small cell lung cancer carcinoma

• Kv1.1 silencing decreased the proliferation of HeLa cells

• Demonstrates potential for treatment across a range of different types of cancers

Why are Dendrotoxins Unlikely to Be Used Therapeutically in Cancers, Where Kv1.1. is Implicated?

• toxins unlikely to be a treatment → something smaller is required to enter cells and have an effect

• Kv1.1. channels are widely expressed in nearly all cells throughout the body

  • While overexpressed in cancer cells, they are expressed in normal cells; treatment would leave these non-viable if targeted

• High expression correlates with poor prognosis in cervical cancer patients

What are Key Features of the KAaH1&2 Toxins?

• Venom isolated from Androctonus australis Hector scorpion

• KAaH1: Kv1.1 and Kv1.3 channels

• KAaH2: selective for Kv1.1 only

• KAaH2 inhibits cell proliferation in human cell lines:

  • U87 (glioblastoma)

  • MDA-MB-231 (breast cancer)

• Inhibits glioblastoma cellular proliferation by KAaH2 toxin via the EGFR signalling pathway

What are Key Features of Margatoxins?

• Isolated from Centruoides Margaritus Scorpion

• Selective for Kv1.3 (may have some activity against Kv1.1 and Kv1.2)

• 1nm injected into tumours restricts the growth of the A549 human lung adenocarcinoma mouse model

What do toxins targeting Kv1.1 and Kv1.3 channels suggest about cancer therapy?

• Some selectivity for cancer cells can be achieved by targeting Kv channels

• Therapeutic challenge:

  • Need appropriate delivery and targeting strategies

  • Toxins are mainly models for developing smaller, cell-permeable drugs

• Context-dependent effects:

  • Kv1.1 and Kv1.3 roles vary between different cancers and cell types

  • Involved in different signalling pathways depending on tumour type

• Effectiveness depends on cancer-specific biology

How do Kv1.3 channels regulate the cell cycle via the voltage sensor model?

• Kv1.3 channels regulate membrane potential and cell excitability

• Possess a voltage sensor domain

• Activation of the voltage sensor → interacts with different intracellular proteins

  • Have a channel zone with different proteins associated

• Channel-associated protein complexes influence signalling pathways

• In cancer, if Kv1.3. Channels are overexpression this leads to excessive cellular proliferation

• Different pathways can be used depending on the cancer cell

• Inhibition of Kv1.3 affects the voltage dependency of the membrane (alters the membrane potential), disrupts voltage sensor signalling and modifies intracellular pathways → reduces proliferation

How does Kv1.3 activity regulate cell proliferation via membrane potential and Ca²⁺ signalling?

• Kv1.3 channels mediate K⁺ efflux, causing membrane hyperpolarisation

• In normal cells there is balanced Kv1.3 activity → controlled membrane potential and activation

• In certain cells, e.g. Cancer cells there is an overexpression of Kv1.3 leading to an increase in K⁺ efflux and hyperpolarisation

• This alters Ca2+ entry into the cell → increased CRAC signalling and cell proliferation

  • Enhanced Ca²⁺ entry via CRAC channels → increased cell proliferation

• Inhibition of Kv1.3 decreases K+ efflux and hyperpolarisation, CRAC-mediated Ca²⁺ signalling and proliferation

• Suggests a dual mechanism is present: membrane potential + Ca²⁺ signalling

How are Kv1.3 Cells Implicated in Apoptosis?

• Kv1.3 is expressed in mitochondria

• Toxins can pass through the mitochondria and bind to Kv1.3

  • Targeting mitochondrial Kv1.3 can trigger apoptotic cell death

• Many toxins cannot cross the cell membrane

• Therefore, they cannot reach mitochondrial Kv1.3 → do not effectively induce apoptosis

• If small, membrane-permeable chemical molecules could pass through the membrane and bind to Kv1.3 channels in the mitocondria they could induce apoptosis in cancer cells

How Are Kv1.3 Channels Involved in Immune Function?

• Kv1.3 channels are important in certain immune cells

• Certain autoimmune disorders are associated with an overexpression of these Kv1.3 cells → potenital target

• Human effector memory T-cells (T_EM cells) play a crucial role in autoimmune disease e.g.

  • Multiple sclerosis

  • Type 1 diabetes

  • Rheumatoid arthritis

• T_EM cells increase in number due to chronic stimulation (of different immune pathways) in these disease states

• Kv1.3 becomes upregulated

Which toxins have been shown to be effective as immunomodulators?

• ShK-L5

  • Derivative of ShK isolated from Stichodactyla heianthus

  • Synthetic shortened version of toxin

  • Decreases CCR7^- effector T cell proliferation

  • Decreases cytokine production

  • Does not affect CCR7⁺  effector T cell (effect is specific toKv1.3 overexpression in CCR7- cells)

  • No global immunosuppression

  • Can target particular effects without removing the immune response

• Margatoxin

  • Reduce cell proliferation of T lymphocytes in cell culture and animal models

What is Cobra Neurotoxin?

•  7000Da peptide isolated from Naja naja atra (Snake venom)

• Acts at multiple receptors but has a high affinity for adenosine receptors -> investigated as an alternative analgesic

• Adenosine receptors are important in mediating nociception/antinociception

What is Cobra Neurotoxins Mechanism of Action?

• At:

  • A₁R – CNT agonist = analgesic effect

  • A_2AR – CNT agonist = hyperalgesic effect

• These receptors have different (& opposing) signalling mechanisms → compound can have both analgesic and hyperalgesic effects

What do hot plate test results show about the analgesic effects of cobrotoxin (CNT)?

• Hot plate test (thermal algesia): measures central (supraspinal) analgesia

  • Endpoint = paw withdrawal latency (pain response)

    • ↑ withdrawal time → ↑ pain threshold (analgesia)

    • ↓ withdrawal time → hyperalgesia

• Effects and responses vary depending on the receptor subtype (A1 vs A2) activated and the dose administered

• High dose CNT acts via A2 receptors and can cause hyperalgesia (increased pain sensitivity)

How does cobrotoxin (CNT) produce analgesic vs hyperalgesic effects?

• Effects depend on adenosine receptor subtype activation (A1 vs A2)

• CNT activates adenosine receptors, mediated by ROS generation and leads to mitochondrial damage

• Driven by breakdown of adenosine into ATP → fuels process

• A1-specific agonist: A1 receptor activation and analgesia

• A2a receptor activation: alters protein production and hyperalgesia

• Demonstrates the importance of correct dosing (dose dependent effects) to ensure analgesic effects to prevent ROS production