P2X7 receptors

P2X7. inflammatory disease. neurodegeneration. cancer/tumour environment. knockout models. arthritis. neuropathic pain. A-740003. disease models. JNJ-42253432. JNJ-54175446. ATP trial.

classification of ion channels

ligand gated:

• activated by extra- or intracellular ligands.

• P2X - example of ATP gated ion channel from outside.

• calcium activated potassium channels - activated by calcium binding to cytoplasmic space.

voltage gated:

• muscle and nerve function.

purinergic receptors and tumour microenvironment

increase in ATP outside cell.

extracellular ATP binds to either P2X (ion channel) or P2Y (GPCR) receptors.

ATP and other enzymes can break it down/metabolise ATP back to adenosine.

adenosine taken back up into cell.

CD39 - break down ATP by removing terminal 2 phosphates, to generate AMP.

AMP metabolised by CD73 to generate adenosine - terminates signal.

P2X7 biophysical properties

trimeric receptor assembly with two transmembrane domains, ATP binds between subunits in extracellular domains.

form cation permeable channel - permeable to calcium and sodium.

need higher concentration of ATP for activation - compared to other subtypes.

P2X7 pharmacology

binding sites for antagonists found at separate site to ATP binding in extracellular domain.

DAMP and P2X7

danger associated molecular pattern.

high concentrations of P2X7 are danger signal in body.

P2X7 and inflammatory disease

non-immune cell source of ATP.

getting epithelial cell death.

release of ATP, can activate immune cells.

triggering pro-inflammatory response.

macrophages express P2X7 - release IL1b in response to P2X7 stimulation.

P2X7 in neurodegeneration

release of extracellular ATP.

activating P2X7 and releasing IL1b and IL18.

IL1b and IL18 both processes downstream P2X7 stimulation.

P2X7 in cancer tumour environment

basal P2X7 activation, low levels ATP, and overstimulation - not understood.

high expression of P2X7 in tumour cells causes tumorigenic phenotype so that they proliferate.

overexpression P2X7 in cell line causes it to proliferate, without additional extracellular ATP - addition minimal ATP to cell line kills cells.

tumour dependent.

P2X7 receptor crystal structure

structural insights:

• trimeric receptor assembly with two transmembrane domains, ATP binds between subunits in extracellular domains.

antagonist binding:

• binding sites for antagonists found at separate site to ATP binding in extracellular domain.

• ATP binding pockets between subunits.

• drug binding pockets between ATP binding pockets - non-competitive antagonists.

crystal structure has no intracellular domains.

has N and C terminus.

had to remove N and C terminus to get crystal structure - partial structure.

P2X7 mouse knockout models

LacZ-neomycin cassette - exon 1 deletion (Glaxo).

neomycin cassette - exon 13 truncation (Pfizer).

LacZ-neomycin cassette - exon 2 and 3 substitution (Lexicon).

short hairpin cassette - exon 3 knockdown (Germal research centre for environmental heath).

CRISPR/Cas9 - exon 2 deletion (Shanghai university).

mouse P2X7 has spliced variants that can escape knockout (e.g. exon 1 alternative and truncated variants).

P2X7 knockout attenuates arthritis

mAbs directed against type II collagen/LPS ip treatment - get inflammatory response.

Pfizer exon 13 truncation knockout mouse.

arthritis score goes up in WT but attenuated in knockout.

reduced percentage limbs affected in knockout mice.

more mice in knockout group have affected limbs.

stained with mAb 9A4 to detect presence collagen cleavage neoepitope (collegen degradation) - reduced in knockout.

stained with sadranin-O (measure of proteoglycan content) - knockout has more staining (increased proteoglycan border) to protect against depletion.

neuropathic pain - P2X7 knockout

partial ligation sciatic nerve - causes thermal hypersensitivity.

thermal sensitivity test - paws on warm pad (WT take away eventually, hypersensitivity take away quickly).

glaxo exon 1 deletion knockout.

withdrawal latency - how quick take away (lower number is quicker).

WT - decreased withdrawal latency.

lost in k/o - protective against neuropathic pain.

humanised P2X7 mouse model - mice expressing human gene products

replace murine P2X7 with human P2X7.

substitute murine exon 2 with human P2X cDNA (exon 2-13) wild type or human P2X7 carrying polymorphism (Gln460Arg).

using murine promoter to express P2X7.

mouse model expresses human P2X7.

humanised P2X7 mouse model - engraftment human cells into mouse (immunocompromised)

e.g. human cancer xenograft model in mice.

mice often immunocompromised (unless looking at immune response).

P2X7 human vs mouse

different pharmacologies.

slight different sensitivity to ATP.

polymorphisms in receptor - associate with different polymorphisms.

P2X7 agonists

ATP.

BzATP.

NAD+.

P2X7 non-selective antagonists

BBG.

KN-62.

OxATP.

P2X7 selective antagonists

A-438079.

A-740003.

AZ101606120.

CE-224,535.

JNJ-42253432.

JNJ-47965567.

JNJ-54166060.

P2X7 inhibitory antibodies

anti-ayu P2X7 polyclonal antibody (aEPAb).

anti-human P2X7 mAb (clone L4).

anti-murine P2X7 mAb (clone 1F11).

P2X7 inhibitory nanobodies

anti-human nanobody (Dano1).

anti- murine nanobody (13A7).

calcium responses human vs rat P2X7 antagonist

Abet compounds (A-740003).

rat and human YOPRO uptake experiment - inhibitory curves.

YOPRO

ethidium is large MW molecule that integrated into DNA - fluoresces when integrated.

healthy cells - nor much fluorescent signal.

permeabilised cells - ethidium/YOPRO moves into cell, intercalated DNA, and fluoresces.

activated P2X7, rapidly triggers membrane permeabilisation - measured on plate reader.

rat and human - inhibition of receptor.

inflammatory pain in vivo models

paw withdrawal latency with carrageenan and CFA - A-74003 (antagonist).

carrageenan/CFA alone - 2 second paw withdrawal latency pause.

carrageenan/CFA + antagonist - reversal of 2 second pause.

spinal nerve ligation.

vehicle/control - fast paw withdrawl - indicative of neuropathic pain.

antagonist - reversal of effect.

P2X7 in tumour growth

inhibition reduces cancer growth in different models.

tumour growth increased in P2X7 deficient mice.

increased growth due to reduction immune cell infiltration into tumour (P2X7 -/- mice versus WT).

hypothesis - P2X7 deficiency reduces immune cell detection of tumours/migration.

P2X7 -/- mice - xenograft tumour expressing P2X7.

tumour recruits immune cells from mouse.

immune cells P2X7 deficient.

selective knockout - just immune cells.

bone marrow transplant shows haematopoietic transfer WT P2X7 mice into P2X7 -/- mice restores near normal anti-tumour response.

P2X7 receptor regulation of tumour immune cell recruitment - melanoma cell line experiment

melanoma cell line - expresses P2X7.

only immune cells P2X7 deficient.

comparing WT and k/o.

analyse post-inoculation.

P2X7 -/- mice:

• increase in tumour volume.

• decrease CD8 and Teff but increase Treg.

• increase CD73/CD39 expression.

• decrease inflammatory cytokine mediators (IL1b and IFNy).

• environment overall looks anti-inflammatory.

• hypothesis/prediction - should decrease ATP in tumour.

k/o decrease in ATP at tumour - explained by protein expression results.

P2X7 receptor regulation of tumour immune cell recruitment - luciferase expressing cell experiment

luciferase expressing cells - detected ATP and fluoresces.

found in fireflies - fluorescence is ATP.

detect with light - image mice to look at light emission.

emits photons.

everything in P2X7 deficient - blocking receptor on tumour and immune cells.

injected into mouse and treated with P2X7 antagonist (A-740003).

decrease in tumour volume - opposite to other experiment.

increase CD4 and effector T but decrease Treg.

decrease CD39/CD73 - metabolising enzymes.

decrease IL1 - P2X7 triggers IL1.

increase IFNy.

hypothesis/prediction - should increase ATP in tumour.

treatment with antagonist - no detection change in ATP (could be bacuse change in ATP release or change volume).

P2X7 receptor regulation of tumour immune cell recruitment

host P2X7 deficiency (including immune cells) increases tumour size, Treg infiltration, and CD39/CD73 - decreases tumour ATP levels.

detect with luciferase experiments.

P2X7 inhibition (A-740003) of host and tumour cells decreases tumour size, Treg cells, and CD73/CD39 - not change in tumour ATP levels.

something compensating or changing system.

JNJ-42253432

abet not good brain penetration.

increasing evidence immune system and inflammatory response involved in array of neurodegenerative diseases and depression - antagonist to cross BBB.

JNJ tested with patch clamp - need lots ATP.

JNJ inhibit human P2X7 in 1321N1 cell line (astrocytoma cells - don’t normally express P2X7 receptors so no background).

took whole blood and peripheral blood monocytes to see if block inflammatory responses - found they could.

inhibition of IL1b release - expected.

JNJ-42253432 brain occupancy

subcutaneous injection - increase in plasma in brain and occupancy along the same timeline.

oral dosing - brain lacking behind (in terms concentration) but sees occupancy as well.

antidepressant trial with JNJ-54175446.

anti-inflammatory approaches proposed for depression treatment.

P2X7 receptors proposed play role in depression pathogenesis based on mouse studies using P2X7 -/- mice with anti-depressant-like phenotype.

CNS-penetrant P2X7 antagonists demonstrated efficacy in chronic stress models.

increase in brain IL1b release (downstream P2X7), P2X7 mRNA upregulation, and microglial activation in chronic stress mice model.

chronic stress linked depression, hypothesised could be good target for depression treatment.

led to ATP trial studying treatment major depressive disorder (MDD) with symptoms resistant to traditional antidepressant drug therapy.

ATP trial

CNS penetrant JNJ-54175446 tested in trial.

multi-centre, double-blind, randomised, placebo-controlled, parallel group trial of JNJ-54715446 with DSM-5 diagnosis of MDD.

moderate severe depression despite ongoing treatment with conventional antidepressive drug.

biomarker profile predictive of activated P2X7 receptor:

• IL1b release.

• inflammatory markers.