28 - IL6 Signalling and Inflammation

Question 1

What is metabolic syndrome?

Answer 1

Metabolic, or cardio-metabolic, syndrome refers to a cluster of related risk factors for cardiovascular disease (CVD) that includes:
o Abdominal obesity
o Diabetes
o Hypertension
o Elevated cholesterol

Question 2

What can metabolic syndrome lead to?

Answer 2

This can lead to serious CVDs, including atherosclerosis

Question 3

How many adults have metabolic syndrome?

Answer 3

A quarter of the world’s adults have metabolic syndrome

Question 4

What is the likelihood of a heart attack in people with metabolic syndrome compared to those without?

Answer 4

Patients are twice as likely to die from, and three times as likely to have a heart attack or stroke compared with people without the syndrome

Question 5

How likely are people with metabolic syndrome to develop diabetes?

Answer 5

People with metabolic syndrome have a five-fold greater risk of developing type 2 diabetes
o Up to 80% of the 200 million people with diabetes globally will die of cardiovascular disease

Question 6

Are there guidelines on treating metabolic syndrome?

Answer 6

The biguanidine, metformin, is often used as the initial diabetes medication but there are currently no established guidelines on treating metabolic syndrome patients with metformin in the absence of overt diabetes

Question 7

What restrictions have been placed on drugs that are used to combat metabolic syndrome?

Answer 7

Use of drugs to combat metabolic syndrome is currently problematic
o Restrictions have been placed by the U.S. FDA on the use of thiazolidinediones due to a reported increase in the incidence of heart attacks and other adverse cardiovascular events

Question 8

Issues with emerging therapies for metabolic syndrome

Answer 8

Emerging therapies (e.g., CB1 antagonists, GLP-1 analogues and 11-beta-HSD1 inhibitors) either have adverse effects or treat only single aspect of metabolic syndrome

Question 9

Class I cytokines (interleukin 6 family)

Answer 9

o IL-11
o Oncostatin M
o Protypical member IL-6

Question 10

Atheroslerosis

Answer 10

IL-6 accumulation in arterial lesions amplifies early stages of atherogenesis by stimulating induction of adhesion molecules and pro-inflammatory cytokins in vascular endothelial cells

Question 11

Vascular endothelial cells (VECs)

Answer 11

Major cellular target for many pro- and anti-inflammatory cytokines
o Activated VECs, SMCs, monocytes and macrophages are each capable of producing IL-6

Question 12

What happens to IL6 in old age, obesity and type 2 diabetes?

Answer 12

In old age, obesity, and type 2 diabetes there is increased expression of the soluble IL6 receptor (sIL-6R)

Question 13

Steps of IL6 expression

Answer 13

1. siL-6R docks with GP130 (found on all cells)
2. recruitment of JAKs (RTK)
3. JAKs phosphorylate STATS (family of transcription factors)
4. STATS interact with target genes (e.g., inflammatory genes like MCP1)
5. monocytes and immune cells attach to vascular endothelium and stimulate transcription
6. other immune cells recruited
7. to resolve signalling, SOCS4 blocks action of JAK by negative feedback to stop further activation of STAT transcription factor

Question 14

Where are STATS normally found?

Answer 14

 STATS are normally found in the cytoplasm, but when IL^ receptors are activated it becomes phosphorylate by the JAK complex, and they dimerise and enter the nucleus

Question 15

What happens with inflammatory signalling by IL6 in metabolic syndrome?

Answer 15

In serious metabolic syndromes we no longer get this resolution of signalling, meaning we get enhanced inflammatory signalling

Question 16

What strategies have been implemented to inhibit IL6 signalling?

Answer 16

A number of ways have been tried to block IL6 signalling:
o Monoclonal antibodies which are known to interact with IL6 and block the interaction with its receptor
o Blockers of the sIL-6R have been trialled
o Monoclonal antibodies blocking the IL6 receptor
- Prevent IL6 from docking
- Used to treat COVID19
o Intracellular blockers
- Inhibitors of JAK tyrosine kinases

Question 17

Examples of IL6 blockers & what do they affect?

Answer 17

• Sirukumab
• Olokizumab
• Clazakizumab
• ALX-0061
• EBI-029
  They affect IL-6

Question 18

Example of a trans-signalling blocker and what does it affect?

Answer 18

FE999301 & it affects siL-6R

Question 19

Examples of IL-6R blockers & what does it affect?

Answer 19

• Tocilizumab
• Sarilumab
• Siltuximab
• ALD518
• NI-1201
  & it affects IL-6R

Question 20

Examples of signalling blockers & what does it affect?

Answer 20

• Tofacitinib
• Ruxolitinib
• CpG-stat3-siRNA
  & if affects specific cell responses

Question 21

The importance of EPAC proteins

Answer 21

o Two isoforms EPAC, EPAC1 and EPAC2
o Directly activated by cAMP in a PKA-independent manner
o Function as GEFs for the Ras-like small GTPase- Rap1

Question 22

Structure of EPAC proteins?

Answer 22

cAMP interacts with the pocket region in the regulatory subunit f EPAC1
o this causes a conformational change in the regulatory subunit
o release of two finger like helices which can interact with Rab1 which opens up the channel for Rap1 to bind GTP and become active

Question 23

How many types of EPAC?

Answer 23

Two types of EPAC:
o EPAC1 which is found in vascular endothelial scells
o EPAC2 is found in pancreatic beta cells and it promotes insulin secretion in animals and in cell culture

Question 24

What does EPAC2 promote?

Answer 24

PAC2 promotes insulin secretion from pancreatic beta cells in vitro and in viv

Question 25

What was the effect of mice lacking EPAC2?

Answer 25

Mice lacking EPAC2 have markedly reduced cAMP-potentiated vesicle/membrane fusion events during glucose-induced insulin exocytosis
o EPAC2 null mice are also prone to obesity and leptin resistance

Question 26

What does sulphonylureas do?

Answer 26

Sulphonylureas have been suggested to exert part of their insulin secretagogue effects through direct interaction with and activation of EPAC2 in pancreatic beta cells

Question 27

How can type 2 diabetes therapies be used in other applications?

Answer 27

Since inhibitors of incretin degradation (dipeptidyl 4 inhibitors) and incretin analogues, e.g., glucagon-like peptide 1 (GLP-1) are currently used for treatment of T2D, new therapies based on novel activators EPAC1 and/or EPAC2 may well have similar therapeutic efficacies

Question 28

What happens when glucose is elevated in the body?

Answer 28

1. Glucose uptake in pancreatic beta cells
2. Increase in ATP which blocks calcium channels
3. Calcium input into the pancreatic beta cells through voltage dependent calcium channels is triggered
4. Insulin secretion

Question 29

What does EPAC2 mediate in the gut?

Answer 29

EPAC2 mediates the actions of gut secretin hormones so when food is ingested, GLP1 and GIPs are released from the gut, and they travel through the bloodstream to the pancreas
o Here they bind to G protein couple receptors of the cell surface and cAMP is elevated
o EPAC2 is then activated, and through a number of mechanisms, including activation of phospholipase C epsilon which produces IP3
o Release of intracellular stores of calcium
o Along with calcium input from the extracellular domain

Question 30

What does the activation of EPAC2 potentiate?

Answer 30

This potentiates the amount of calcium released therefore there is enhanced insulin secretion
o Enhanced vesicle trafficking and exocytosis

Question 31

EPAC1

Answer 31

o EPAC1 is expressed at low levels in human islets, and knockout of EPAC1 gene expression is also linked to glucose intolerance in mice
o Studies indicate multiple “protective” roles for EPAC1 in the vascular endothelium

Question 32

What does EPAC1 exert?

Answer 32

EPAC1 exerts multiple anti-inflammatory actions in vascular endothelial cells

Question 33

What happens when EPAC1 is activated?

Answer 33

When EPAC1 is activated, it activates Rap 1 which leads to the induction of the SOCS3 gene to block IL6 signalling in vascular endothelial cells
o It does this by blocking JAK tyrosine kinases and STAT3 activation

Question 34

What is EPAC1 involved in?

Answer 34

Involved in other signalling events involved in inhibiting inflammation, so for agents that increase permeability or leakiness of vasculature which is normally involved in inflammation

Question 35

How does EPAC1 stop inflammation?

Answer 35

o Activation of EPAC1 blocks that by interactions with the cell cytoskeleton and promoting cell:cell contacts by cadherins and cell basement interactions by activation of integrin receptors
o So EPAC1 will caused increased stickiness of the endothelial cells to the basement memebrane and block leakiness of the vasculature by strengthening cell:cell contacts and preventing immune cell entry

Question 36

New ligands to activate EPAC1 and EPAC2

Answer 36

Generation of analogues of cAMP which first target EPAC1 but not 2

Question 37

Role of EPAC1 in the inhibition of IL6 signalling

Answer 37

o IL6 is causing phosphorylation and activation of STAT3
- Blocked when you activate EPAC1

Question 38

Role of EPAC1 and Rap1 in SOCS3 induction

Answer 38

o If you knockout EPAC1 in vascular endothelial cells with siRNA you knockout expression, that blocks SOCS3 induction which shows that if you activate EPAC1 with cAMP that increases SOCS3 production
o If you knockout Rap1 with siRNA, expression is reduced showing that is involved in SOCS3 induction

Question 39

Difference between MAP kinase-DEPENDENT SOCS3 induction and MAP kinase-INDEPENDENT SOCS3 induction?

Answer 39

DEPENDENT involves JNK and ERK whereas INDEPENDENT involves EPAC1 and FLAVANOIDS for transcriptional control

Question 40

SOCS3 induction by EPAC1 - drugs and agents

Answer 40

o Current cAMP-elevating agents (e.g., forskolin) are not suitable EPAC1 activators because the global elevation of cAMP they achieve leads to the activation of EPACs PLUS protein kinase A
o Currently cAMP based anti-inflammatory drugs (e.g., the FDA approved PDE inhibitors, Apremilast and Roflumilast) are efficacious as anti-inflammatory agents but display side effects (typically nausea, emesis, diarrhoea, and arrhythmia)
o 8-CPT-2Me-cAMP is also unable to discriminate between EPAC1 and EPAC2, and therefore activates each isoform equally well resulting in arrhythmia and reduced cardiac function in animal models through EPAC2

Question 41

Rational

Answer 41

o We envisage administration for a single, EPAC1 selective activator that could simultaneously treat multiple aspects of metabolic syndrome, including chronic inflammation and insulin resistance, which would otherwise require multiple agents
o These new compounds will either complement, or improve on, the beneficial actions of existing anti-diabetic drugs such as metformin
o We predict that EPAC1 activators would exert largely positive effects in patients with metabolic syndrome and therefor de-risk drug development

Question 42

Synthesis of EPAC1 protein

Answer 42

We synthesised some EPAC1 protein
o Synthesized the cAMP binding domain of EPAC1 along with EPAC 2
o These were purified with bacteria to show that they could bind cAMP in a test tube
fluorescent cAMP binds more to EPAC2 and EPAC1
o shows these proteins are folded

Question 43

Binding assay of EPAC1

Answer 43

o The methylate forms of cAMP binds better, but the best form is 8-CPT
o We can use this binding assay to screen for compounds that could compete out the binding of cAMP to these proteins and identify novel molecules

Question 44

HTS (high throughput screening) for cAMP competitors on PAC1 CNB

Answer 44

o 500 compounds were screened to develop the assay, then a further 1500 compounds were screened which had known therapeutic benefits and measured their ability to compete the cAMP binding to the regulatory domain of EPAC1
o Compounds in red- effective and reproducible inhibitors of cAMP binding
o 300,000 compounds were screened in this assay using robotised mechanisms and identifying some binders

Question 45

What can you do in more refined assays for cAMP binding?

Answer 45

In more refined assays, you can do competition assays with cAMP
o We found that compound I942 was the better inhibitor of cAMP binding

Question 46

Compound I942 in as EPAC1 activator

Answer 46

o We showed that I942 compounds actually activates EPAC1 in the test tube
o And we could model the binding of this compound to the cAMP binding domain and determine its mode of binding

Question 47

Future directions?

Answer 47

We have identified I942 as a first-in-class, non-cyclic nucleotide EPAC1 partial agonist
o We will now:
- Undertake bespoke series expansion of I942, supported by in silico models supported by biophysics, to establish the key structure determinants that can be exploited for affinity and efficacy gain
- We will focus on optimising target potency and cell efficacy with the further aim of obtaining oral bioavailability, good pharmacokinetics and minimising potential toxicity
- We will then progress a lead series into EPAC1 animal models of CVD, obesity and T2D