Selective Neurotoxins and their uses

define venomics

the isolation of individual toxins within venoms to be analysed and studied

discuss how venoms and toxins have been used to aid scientific research

• Venoms can contain around 40-50 different components each of which can target particular areas of the physiological system- high selectivity

• Neurotoxins e.g. can target ion channels such as K+/Na+/Ca2+, nAchR, NMDAR, ASICs, to enhance or inhibit activity

• used to study ion channels understand their role, structure, binding sites, point mutations, gating characteristics

• also been able to precipitate and isolate ion channel to help identify the sub families of channels that are available

• Many drug development has been created from venom components e.g. ACE inhibitors such as captopril

describe the structure and characteristics of toxins

structure

• Small peptides but can vary in length between 8-70 aa

• Compact small scaffold structure

  • alpha and beta sheets give it compact 3D structure

  • stabilised by S-S bridges (usually 2-4) and H-bonds

• characteristics- High specificity, activity and no accumulation

describe the origin and MOA of mambalgins

origin- black and green mamba 

• MOA- they inhibit ASICs by binding to a particular binding site in the e/c acid sensing part of the channel

describe the structure, subunit localisation and activation mechanism of mambalgins

structure

• Mediate a Na+ selective current, but can also let other cations through the channel

• 4 members- 1-4

• exist as homo or heterotrimers

subunit localisation

• ASIC1a, ASIC 1b, ASIC2a, ASIC2b, ASIC3 expressed in sensory neurones in the PNS

• ASIC1a, ASIC2a, ASIC2b, ASIC4 widely expressed in the CNS

activation

• H+ binding to e/c surface leads to channel pore opening and influx of ions. This leads to excitation and activation of the pain pathway

describe the 3- finger toxin formation and their characteristics

• 3FT formed due to 4 S-S bridges

• 3FT exist as different families one of which is ASICs- have different properties and activities

• mambalgins belong to new 3FT as they have different structure- beta strand loops I and III are shorter than loop II

by comparing mambalgin to morphine, discuss experimental evidence for the efficacy of mambalgin 1 as analgesic

• currently no good medications for chronic pain

• current gold standard opioids have limited use due to high abuse liability

• acute inflammatory pain model used to measure effect of morphine, mambalgin and PcTx on paw withdrawal threshold when administered intrathecally

• morphine increase pain withdrawal- effective

• mambalgin 1 not as effective as morphine but produces significant analgesic effect and is non-opioid dependent (not blocked by naloxone)

• effect of morphine decreases over days due to tolerance

• less tolerance with mambalgin- more sustained analgesic effect over time and minimal respiratory depression

• ASIC1a -/- loses analgesic effect of mamalgin 1

• similar effects seen with intraplantar admin (into paw) but mamalgin 1 analgesic effect maintained with ASIC1a -/-

• suggests working through different ASIC subtype

describe the origin and MOA of dendrotoxin

black and green mamba

MOA-  they block Kv channels leading to increased synaptic activity

• Leads to actional potential broadening, slowing of membrane repolarisation and increased neurotransmitter release

• DK from black mamba selectively blocks alpha subunit of Kv1.1 channel

describe the role of Kv1.1 and 1.3 channels, specifically in the context of cell growth

• Control membrane excitability, cell volume regulation modulation of intracellular K+ concentration

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

• This is important in G1 to S phase of the cell cycle where the membrane excitability is important for preparing the cell for the growth phase

• It regulates cell volume via the efflux of K+ and that allows shrinkage of the cell, which is important for preparing the cell for growth

using experimental evidence discuss the therapeutic benefit and limitations of dendrotoxin K (DK) in treating cancer

• Kv1.1 overexpressed in some cancer types

therapeutic benefit of DK

• disrupt cell growth in tumour cell lines and mouse tumours

• arrests the cell cycle by disrupting G1-S transition stage only when injected directly into tumours

• Increases protein expression of the cyclin dependent kinase inhibitors e.g. p27Kip1

• decreases protein levels of cyclin D3

experimental evidence

• DK causes visible reduction in tumour volume

• 10nM dendrotoxin can reduce cell proliferation of NCF-7 breast cancer cell line cells by 30%

• 100nM dendrotoxin has been shown to reduce cell proliferation in chemoresistant NSCLC carcinoma

• silencing Kv1.1 decreases proliferation

• high Kv1.1 expression correlates with poor prognosis

limitations

• cannot be used as treatment as we need something small to enter cells

• Kv1.1 ubiquitously expressed so selectivity is hard

describe how Kv1.3 is implicated in cancer and autoimmune disorders

cancer

• If overexpressed it changes the way calcium enter the cells and that leads to excessive proliferation, so blocking it will reduce Ca2+ entry and proliferation

• Kv1.3 also implicated in apoptosis but mainly via mitochondrial Kv1.3 channel

immune disorders

• Certain autoimmune disorders have overexpression of Kv1.3 cells such as MS, T1D, RA

• leads to chronic stimulation in these disease states which causes an increase in number of human effector memory T cells

describe the dual actions of cobra neurotoxin, and use experimental evidence to supplement this

• has high affinity for adenosine receptors- important for mediating nociception and antinociception

  • agonist at A1R- analgesic effect

  • agonist at A2AR- hyperalgesic effect

• mechanism

  • Under cellular stress, ROS are produced and this can damage mitochondria

  • Since adenosine is made from mitochondrial ATP this disruption increases adenosine signalling which then activates its receptors

  • Cobra neurotoxin acts like agonist at A1R leading to analgesic effect but also activate A2AR and that triggers signalling pathways such as MAPK/ERK, altering protein production

• experimental evidence

  • Hot plate test measures central analgesic effect via paw withdrawal threshold

  • Different effect seen with low or high doses of the cobra neurotoxin- at high dose we get prominent A2AR activity leading to hyperalgesia rather than analgesic

give an example of a venom/toxin that can be used to target immune disorders

ShK-L5

• synthetic derivative of ShK

• Decreases CCR7^- effector mediated T cell proliferation and cytokine production

• However it does not affect CCR7⁺ effector T cell so we don’t get a global immunosuppression; means we can target particular effects without removing immune response completely