Ionotropic receptors: therapeutics Flashcards
(33 cards)
What is the pathophysiology of epilepsy?
Imbalance in Glu/GABA transmission → hyperexcitability
Synchronous bursts of APs (firing APs constantly causes loss of signal-noise ratio)
Status epilepticus can lead to excitotoxicity
iGluRs may play role in triggering seizure (triggering may involve loss of GABA activity?)
iGluRs involved in actual seizure activity (definitely involved in synchronising activity / passing AP bursts between neurons)
What are the current mechanisms for licensed epilepsy treatments?
Potentiate GABA synapses - (diazepam)
Inhibit VGSCs (particularly blockers that have use-dependent effects i.e. block channels that are more active) - (phenytoin)
Inhibit VGCCs (ethosuximide)
Activate VGPC - (retigabine)
Inhibit NDMARs: FELBAMATE
Inhibit AMPA: PERAMPANEL
How does felbamate work?
Not entirely understood but seems to be NAM of both GluN1 and GluN2 receptors
*also PAM of GABA-A receptors
How does perampanel work?
AMPA NAM (binds between LBD and TMD)
- not selective for any subtype of AMPAR
- only licensed for PARTIAL seizures in UK
- side effects: somnolence, dizziness, fatigue, falls - if blocking AMPA across brain, will get widespread side effects e.g. cerebellum - decreasing balance
What could provide a potential new approach for treating epilepsy? What evidence has been used to suggest this method?
Targeting AMPARs that are associated with Ɣ-8
Forebrain regions important in epilepsy (cortex / hippocampus / temporal lobe)
mRNA expression used to indicate TARP expression in mouse/rat (although less accurate as may not be a linear relationship between them):
Ɣ-2 very widespread (avoid)
Ɣ-3; forebrain but also other areas
Ɣ-4; not really expressed in cortex/Hi
Ɣ-8; more selective in cortex & hippocampus, less in other areas
What are TARPs involved in?
Traffic AMPARs to surface (e.g. KO Ɣ-2 - lose AMPARs in cerebellar cells)
Ɣ-2, Ɣ-3, Ɣ-4, Ɣ-8 interact with AMPA receptors
(the other 4 interact with calcium channels)
Change glutamate affinity, desensitisation, deactivation & pharmacology - effectiveness of some drugs is changed by TARP expression
What is a FLIPR assay?
Fluorescence Imaging Plate Reader
Common cellular assay in drug development (screening)
Grow cells (HEK / Chinese hamster ova etc.) which express protein of interest in plate with 384 wells - calcium sensitive fluorescent dye loaded into cells - add as solution then wash off excess
Machine can add many drugs + measures fluorescence -can get responses from all wells in 6 mins
First give standard conc of each drug to screen compounds, then refine the compound (add groups / substitutes etc.)
How has FLIPR been used to find drugs selective for Ɣ-8-associated AMPARs?
(Kato et al)
Expressed GluA1(o) in cells with TARPs, and used GYKI (NAM) as control (inhibits response in all cells)
Screened 1000s of compounds for differentiation between Ɣ-2 & Ɣ-8 expressing cells
LY3130481 (antagonist) = conc-dependent inhibition of AMPA response: greater inhibition with Ɣ-8 than Ɣ-2. Also went on to show selective inhibition (Ɣ-8) in flip + flop GluA1/2/3/4
BUT, cultured cells, not relevant to real neurons
After testing with FLIPR (cultured cells), what was LY3130481 tested on?
Rat hippocampal slice: add glutamate every 30s, then add increasing concentrations of drug - AMPA response inhibited - get IC50
Still potent (although slightly less potent than in cultured cell lines)
How was it demonstrated that LY3130481 is dependent on Ɣ-8 in real neurons?
Mouse hippocampal slice: Ɣ-8 KO
No inhibition of AMPA response in KO
In KO: still get other AMPARs going to the surface - but other TARPs
How was the potential role of LY3130481 in epilepsy tested? What did this show?
Inducing seizures in animals: pre-treat with drug then measure % that had convulsions
Vehicle: 100% had seizures
LY3130481 acted as anticonvulsant in animal model
*More potent than GYKI or perampanel
Why is LY3130481 better than GYKI / Perampanel?
- Given orally as crosses BB-barrier (GYKI and perampanel = I.P. injections, may not cross as well)
- More potent (GYKI/perampanel needs higher concentrations to stop seizures)
- No motor impairment - actually shows non-significant increase in motor function (perampanel impairs motor function at doses with anti-epileptic activity as inhibits AMPARs across the brain)
What is the exact mechanism of LY3130481?
Directly binds to Ɣ-8 TARP, even in the absence of AMPAR subunits (shown by crystal structures)
Not yet known how it changes AMPAR function..
Outline pathophysiology of stroke
Majority: ischaemic (artery thrombosis) Remainder: haemorrhagic (cerebral artery rupture) - CT scan needed to diagnose
Three key processes in ischaemia:
1) Inflammation (cells die/burst → release inflammatory mediators)
2) Free radical production
3) Rise in extracellular glutamate concentration (causes excitotoxicity - persistent neuronal activation and NMDARs activated)
How is ischaemic stroke currently treated?
Tissue plasminogen activator (tPA) within 3 hours
- Only for ischaemic, link with functional benefits in patients
- If >3 hours, restoring flow can spread free radicals to other areas causing more damage
What are the key elements in the cascade leading to cell death in stroke (necrosis vs apoptosis)
1) Na⁺ + Ca²⁺ influx upsets osmotic balance → NECROSIS → cell lysis & release of contents including glutamate & inflammatory mediators
Calcium influx also causes..
2) Mitochondrial impairment → less ATP → free radical production (oxidative stress) → CYTOCHROME C release (APOPTOSIS initiator)
3) Glutamate release from presynaptic terminals
Finally..
4) REVERSAL of glutamate TRANSPORTERS causes even more glutamate release!
(due to loss of function of Na⁺/K⁺ exchanger which usually maintains physiological Na⁺/K⁺ concentration
What steps result in Ca overload in stroke?
Glu activates AMPARs - removes Mg²⁺ block of NMDARs: rise in [Ca²⁺]i
This depolarisation activates VDCCs, further increasing [Ca²⁺]i
Activation of mGlu1 or 5 also increases [Ca²⁺]i
Internal Ca²⁺ stores e.g. endoplasmic reticulum and the Ca²⁺ efflux pump cannot cope with rise in [Ca²⁺]i
*resting calcium inside cell usually ~100nM tightly controlled by efflux pump and pump into stores like endoplasmic reticulum
How does Ca overload cause cell death in stroke?
Mitochondria take up Ca²⁺, this causes uncoupling of ATP production (less ATP) and formation of reactive oxygen species (ROS - can lead to cell death)
Ca²⁺ dependent enzymes activated indiscriminately leading to cell death:
- lipase;
- protease;
- endonuclease; break down DNA
- phospholipase A2
- nitric oxide synthase
How do free radicals contribute to stroke pathophysiology?
Mitochondria produce Superoxide (O₂⁻):
highly reactive + produces other free radicals → neuronal death
- SOD (destroys superoxide) cannot cope with excessive production during ischaemia
- nNOS is activated by Ca²⁺ and produces free radical nitric oxide (NO*)
- Inducible NOS (iNOS) activated in response to inflammation: produces much more NO* than nNOS
NO* can react with superoxide (O₂⁻) to form peroxynitrite which damages DNA and proteins, is membrane permeable so spreads damage to neighbouring neurons
In stroke, what causes an increase extracellular glutamate? And what is the effect of this?
Activation of PLA₂ by Ca²⁺ causes ARACHIDONIC ACID production, which inhibits glutamate transporter and produces free radicals
*Activation of NMDARs, AMPARs and KARs increases [Na⁺]i, this leads to NECROSIS
Calcium also disrupts mitochondria: low [ATP] causes Na⁺/K⁺ exchanger failure→ disruption of Na⁺/K⁺ ion balance causes reversal of EAAT (starts to pump out Glu)
What is the anti-excitotoxic hypothesis? (stroke)
Protect neurons by immediate treatment with a GluR antagonist or Glu release inhibitor
What possible anti-excitotoxic drugs for stroke have been looked at?
NMDAR antagonists:
- shown to reduce extent of brain lesions (neuroprotective) in animal models of ischaemic stroke (e.g. tie off cerebral/carotid artery- give drugs, then relieve tension so that blood flow returns)
Competitive NMDAR antagonists, channel blockers, glycine site antagonists & NBQX (AMPAR antagonist) all failed in clinical trials
What effects of anti-excitotoxic drugs resulted in ending the clinical trials?
Lack of neuroprotective effect (therapeutic brain concentrations may not have been achieved e.g. BBB crossing issues)
Adverse effects such as hallucinations, effects on memory and ataxia (loss of motor coordination)
Why have NMDAR antagonists failed in clinical trials?
1) Should give within 2 hours, but treatment windows were extended to several hours
2) 6-24 hours post stroke, minor, sustained [Glu] increase may promote neuronal survival by NMDAR-DEPENDENT transcriptional activation of PRO-SURVIVAL GENES
3) Loss of WM (axons) + glia: large impact on outcome of ischaemia in humans? GluN2C / GluN2D may play a role in WM injury + 1st generation competitive antagonists were GluN2A/GluN2B selective
4) GluN3 NMDARs are expressed on oligodendrocytes- current competitive NMDAR antagonists may not block these effectively (as some don’t bind glutamate)
5) As NMDARs not the only proteins involved in neuronal cell death -may need combination of different drugs
