References

(1)    Zhao, Q. 2010. Dual Targeting of CCR2 and CCR5: Therapeutic Potential for Immunologic and Cardiovascular Diseases. Journal of Leukocyte Biology 88(1): 41-55

(2)    Struthers, M. and Pasternak, A. 2010. Common challenges to the development of CCR2 agents have included poor activity at the rodent receptor and selectivity for both other chemokine receptors and ion channels. Current Topics in Medicinal Chemistry 10(13): 1278-98

(3)    Proudfoot, A. 2008. Is CCR2 the Right Chemokine Receptor to Target in Rheumatoid Arthritis? Arthritis and Rheumatism 58(7): 1889-1891

(4)    Kang, Y. et al. 2010. CCR2 antagonism improves insulin resistance, lipid metabolism, and diabetic nephropathy in type 2 diabetic mice. Kidney International 78(9): 883-94

(5)    Murphy P. 2008. Janeway’s Immunobiology. New York. Garland Science

(6)    Martinez, H. et al. 2011. Critical Role of Chemokine (C-C Motif) Receptor 2 (CCR2) in the KKAy + Apoe -/- Mouse Model of the Metabolic Syndrome. Diabetologia

(7)    Brodmerkel, C. 2005. Discovery and Pharmacological Characterisation of a Novel Rodent-Active CCR2 Antagonist, INCB3344. The Journal of Immunology 175: 5370-5378

(8)    Chuang, S. et al. 2011. Cilostazol Redusces MCP-1-induced Chemotaxis and Adhesion of THP-1 Monocytes. Biochemical and Biophysical Research Communications 411: 402-408.

(9)    Gilbert, J. et al. 2011. Effect of CC Chemokine Receptor 2 CCR2 Blockade on Serum C-Reactive Protein in Individuals at Atherosclerotic Risk and With a Single Nucleotide Polymorphism of theMonocyte Chemoattractant Protein-1 Promoter Region. The American Journal of Cardiology 107(6): 906-911

(10) Cullen, J. et al. 2007. Resveratrol inhibits expression and binding activity of the monocyte chemotactic protein-1 receptor, CCR2, on THP-1 monocytes. Atherosclerosis 195: 125-133

(11) Cherney, R. et al. 2009. Discovery of trisubstituted cyclohexanes as potent CC chemokine receptor 2 (CCR2) antagonists. Bioorganic and Medicinal Chemistry Letters 19:597-601

(12) Lommen, G. et al. 2005. 2-Mercaptoimidazoles, a new class of potent CCR2 antagonists. Bioorganic and Medicinal Chemistry Letters 15:497-500.

(13) http://clinicaltrials.gov/ct2/results?intr=%22CCR2+Antagonist%22

(14) Matsushima, K. et al. 2011. Chemokines in inflammatory and immune diseases. Inflammation and Regeneration 31(1): 11-21

(15) Gerszten, R. et al. 1999. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 396: 718-723

(16) Zernecke, A. et al. 2008. Chemokines in Atherosclerosis: An Update. Atherosclerosis, Thrombosis and Vascular Biology 28: 1897-1908

(17) Melgarejo, E. et al. 2009. Monocyte Chemoattractant Protein-1: A Key Mediator in Inflammatory Processes. The International Journal of Biochemistry and Cell Biology 41: 996-1001

Current Status and Conclusions

CCR2 is still a long way off being used as a clinical therapy but as I have hopefully demonstrated, it has significant potential, at least for more research to be done into its uses, even if it never appears as a liscenced treatment. Here is a quick summary table of the current status of drugs that are being tested at the moment. For a wider view on the current status of other potential chemokine therapies visit the review that I adapted this table from, at http://www.jsir.gr.jp/journal/Vol31No1/pdf/03_R2_11.pdf

From what I have gleaned from my research, MLN1202 seems one of the most promising drugs. As I have already described, it is a monoclonal antibody that has actually been tested on humans with significant reduction in C-reactive protein (a biomarker for atherosclerosis). For this reason, it seems plausible that once safety aspects have been assured, this drug has the potential to provide clinical benefit to patients with atherosclerosis disease as it has proven efficacy in humans, not just animal models.

Conclusions

I have aimed to give a comprehensive review on CCR2 blockade and hopefully I have provided enough information on the subject so that you can see the significance of this topic but also so that you can make your own mind up about how useful it is to aim chemokine research towards this specific receptor. Whether I have helped you along the path to becoming a budding scientist or not, I hope you found my blog useful in some way or another!

Some Solutions...

The failure of CCR2 blockade to come into the clinic inevitably leads to the assumption that something is going wrong with the transfer from animal models into humans and something must be done to rectify this if CCR2 is to become a successful therapy.

A major solution to this problem that  has been suggested and tested by several research groups is the idea of targeting multiple receptors, in particular CCR5 in conjunction with CCR2. And here is an explanation for why…

Targetting two or more receptors poses some problems when implementing the drug into the clinic, mostly concerning safety. Here are just a few examples of some of the issues raised.

This is just one of the ways of overcoming the current problems. I have barely touched the surface with regards to the extent of research into this topic and there are many hurdles yet to overcome.

The Drugs

Surprisingly, clinical trials of drug therapies aimed at targeting CCR2 receptors have failed. The only drugs targeting chemokine receptors that have been liscensed are those resulting in CCR5 inhibition for HIV/AIDS treatment and CXCR4 blockade for autologous transplantation in non-Hodgkin’s lymphoma¹. This may be due to:

1)       A lack of potency by targeting a single chemokine receptor

2)      Activity of the rodent receptor not correlating with the amount in humans

3)      Selectivity of the antagonist – targeting of other chemokine receptors unintentionally. This leads to questions of safety².

Here is a summary table of potential CCR2 antagonists that have been largely characterised in rodents, with some going as far as being studied in phase II trials. Some I have mentioned in previous posts but hopefully you will find it useful for them to be collaborated in a single place. 

As you can see, there is a vast amount of research going on out there. However, the failure of most of these agents on entering the clinical trial phase in humans, leads to the questioning of how useful CCR2 blockade actually is. For this reason, I will have a brief look at the alternatives out there and perhaps some solutions so that CCR2 can be transferred into clinical use.

CCR2 and Disease: Other Examples

So, as I have demonstrated so far (I hope!), the main action of CCR2 in disease is causing the infiltration of immune cells (mainly monocytes) into tissues where it is not necessary or where the endogenous immune response has become amplified too much so that it results in diease. Consistent with this fact is CCR2’s involvement in other diseases, for which I have created a summary list below. This is by far an extensive list of where CCR2 manifests in disease but hopefully it will give you a feel for the vast array of diseases that this CC chemokine receptor contributes to and also the research that is done as a consequence.

For more information, Janeway’s Imunobiology textbook (8^th Edition) is a good source for the mechanisms which are involved. Greater detail of the antagonist INCB3344 mentioned here can be found in an article published in he journal of Immunology which can be accessed by this link: http://www.jimmunol.org/content/175/8/5370.full.pdf+html

I hope you now have an appreciation for the scope of how important CCR2 research is. To highlight this (and for ease of reading), I will next go on to present a table summarising the CCR2 blocking drugs I have mentioned so far and some new ones that I have found along the way.

CCR2 and Disease: Rheumatoid Arthritis

Rheumatoid Arthritis: Basic Facts

  

Here is a video created by Pfizer that is also useful to gain a little more knowledge about rheumatoid arthritis as well as providing some good visual sources of what the disease looks like.

http://www.youtube.com/watch?v=EPmF4vWkuCk

How does CCR2 contribute to Rheumatoid Arthritis?

The mechanism of how rheumatoid arthritis develops is understood to some extent but there are large areas of uncertainty too. For example, the trigger that sets off this autoreactivity to self in the joints is unknown. However, it is known that the process is dependent on both T and B cells as knock-out mice who are deficient of both these lymphocytes have shown to be resistant to the disease. The diagram below shows the progression though which the mechanism of rheumatoid arthritis was discovered.

It is now known that CD4⁺ T cells contribute to the intiation of the disease and macrophages cause perpetuation of the disease. The animation below shows how this process works.

So, we can see that CCL2 and therefore its receptor CCR2 play a role in the early initiation stages of the disease and also the progression towards a chronic inflammatory state. For this reason, it has been and still is a major topic of research as a therapeutic target. At the moment, the ‘gold-standard’ treatment for rheumatoid arthritis is anti-TNF-α treatment (involving antibodies targeted towards this cytokine) alongside steroids and methotrexate (Disease Modifying Anti-Rheumatic Drugs or DMARDS). Whist this treatment is effective in around 40% of patients, there is still a need to develop new therapies to tackle this disease in patients who are non-responders in cases of secondary and tertiary resistance (1).

Table showing how these potential therapeutic interventions have been studied

This table demonstrates the importance of CCR2 targeting for rheumatoid arthritis treatment although it does highlight some discrepancies as to how effective inhibition can be achieved. Also, it is worth noting that some studies using genetic approaches with knock-out mice have showed contradictory evidence as to whether CCR2 is an effective target for ameliorating this disease. A worsening of arthritis was seen with CCR2-deficient mice which can be explained by activation-induced cell death due to CCR2 deficiency. This raises the issue of safety and where it is plausible to knock-out these receptors in the clinical setting (1).

Is CCR2 the Correct Receptor to Target?

In light of the failings of clinical trials using CCR2 blockade in rheumatoid arthritis, it has to be questioned whether CCR2 is worth targeting in this disease. As shown in the mechanism of the disease profile and the studies done in animal models, CCR2 definitely contributes to rheumatoid arthritis incidence or progression in some way. However, it is now noted that other synovial fluids may be acting as the ligand to CCR2 to induce monocyte chemotaxis, rather than MCP-1/CCL2. This may be why failure of CCR2 therapeutics in rheumatoid arthritis has occurred. It has been suggested that CCR1 is a more useful target. Although it has failed in clinical trials, an increase in receptor occupancy of CCR1 may prove to be successful in vivo (3).

Conclusions

1)      CCR2 is involved in rheumatoid pathogenesis

2)      It has been researched extensively in mice with some successful results using mainly antagonists to the receptor

3)      Clinical trials so far have failed

4)      It may be more feasible to look at targeting a different chemokine receptor in this disease or looking at targeting a variety of chemokine receptors at once

I hope I've shown you how current a topic CCR2 blockade is with regards to rheumatoid arthritis. Whilst many developments have been made in the area of research, it is quite clear that more work needs to be done in order to transfer the findings into the clinic.

CCR2 and Disease: Atherosclerosis

Due to the fact that CCR2 and its ligands are heavily involved in monocyte infiltration, they have been characterised in diseases that have high levels of monocyte infiltrates. This includes atherosclerosis, rheumatoid arthritis, multiple sclerosis, delayed hypersensitivity reactions, bacterial infection, and renal disease. In order to explain the concepts of how and why CCR2 blockade is used for therapies, I am going to look firstly at what the disease is, how CCR2 activation contributes to the disease state and whether targetting CCR2 is beneficial in ameliorating the symptoms. For ease of understanding, I am going to collect all my findings with regards to drugs that are targeted at antagonising CCR2 and present them in a table.

Atherosclerosis

Atherosclerosis is characterised by plaque formation in the blood vessel walls which narrows the lumen and impedes normal blood flow, leading to consequences such as turbulence and blockage resulting in myocardial infarction. These plaques consist of fatty streaks and are a result of the infiltration of monocytes through the blood vessel walls and into the bloodstream. Hence, atherosclerosis is known as an inflammatory disease. MCP-1 contributes to this process by aiding the extravasation process of monocytes into the bloodstream.  Here is a little animation that hopefully will help you to understand MCP-1’s role in atherosclerosis(15).

Basically, the key point to take from this is that MCP-1 is important in turning the rolling leukocyte attachments (initiated by e-selectin binding to sialylated Lewis-X moieties on the monocyte) into firm adhesions so that the monocyte can pass between the vascular endothelial cells and into the lumen of the blood vessel. The MCP-1 is bound to the extracellular matrix of the blood vessel wall along with CXCL8 which also contributes although probably more to neutrophil chemotaxis than to monocyte recruitment (16). Here is an animation to demonstrate this process visually.

For more information on both MCP-1 and IL-8 contribution to atherosclerosis, follow this link to read a ‘Letters to Nature’ Journal on the topic:  http://www.nature.com/nature/journal/v398/n6729/pdf/398718a0.pdf

Why is this relevant to atherosclerosis?

Monocyte entry into arteries contributes to atherosclerotic lesion formation as these monocytes differentiate into macrophages on entering the intima of the blood vessel wall. They can then accumulate and eventually turn into a foam cells. The animation below shows how this is leads to atherosclerotic plaques.

As I have shown, MCP-1 and therefore activation of CCR2 receptors, has been shown to be crucial in the development of atherosclerosis with CCR2 being characterised as the most abundant chemokine receptor on the surface of human blood monocytes (as proved through antibody staining). For any budding scientist, pharmacologist or researcher of drug development this is an obvious signal to suggest CCR2 inihibition as a potential therapy to ameliorate monocyte infiltration and therefore decrease atherosclerosis development.

Looking at CC Chemokine Receptor 2 Function Specifically

Now that I have looked at the basics of what chemokines do in general, I am going to look specifically at the function of CC chemokine receptor 2 and its ligands. Once we know the mechanism of action of this receptor and it’s chemokines and how it functions under normal circumstances we can then go on to look at what its aberrant expression causes and how this can be treated.

Here is a little summary table to describe the properties of CCL2/MCP-1:

Produced By	Monocytes Macrophages Fibroblasts Keratinocytes Vascular Smooth Muscle Cells Endotheleial Cells T cells
Receptor	CC chemokine Receptor 2 (CCR2)
Attracts	Monocytes Natural Killer Cells Basophils Eosinophils Dendritic Cells
Major Effects	Activates macrophages Basophil histamine release Promotes T-helper 2 (TH2) cell immunity

Adapted from Janeway's Immunobiology by Murphy and Travers (2008).

MCP-1 Structure

There are 4 known MCP chemokines involved in human immunity, MCP1-4. MCP-1 is secreted in response to inflammatory signals in two forms that have molecular weights of 9kDa and 13 kDa. The differences in molecular weights are to do with O-glycosylation but this doesn’t affect their ability to attract monocytes and other immune cells. The residues in the amino terminal are crucial in the functioning of the MCP-1 protein. The mains ones include:

• Residues 1-6: contribute to the chemoattractant ability of MCP-1 (Asp-3 in particular has been noted as a key player)
• Residues 7-10: involved in receptor desensitisation
• Amino Acid at Position 1: important in the secondary structure formation of the protein and therefore the binding of MCP-1 to recpetors

It has been most commonly noted that MCP-1 binds to CCR2 in a dimeric fashion, although monomers of MCP-1 have also been known to cause activation of receptors too (17).

Functions of MCP-1

The ability of MCP-1 to cause chemotaxis and the movement of cells is thought to be due to the fact that through the signally pathway, CCR2 activation activates phospholipase C and therefore, eventually increases the concentration of intracellular calcium through IP3 signalling. A schematic diagram is shown to summarise this process along with the other theories of how chemotaxis is enabled: that of NFκB activation and Rho activation.

Adapted from Melgarejo, E. et al. 2009. Monocyte Chemoattractant Protein-1: A Key Mediator in Inflammatory Processes. The International Journal of Biochemistry and Cell Biology 41: 996-1001

MCP-1 Signalling. 

Cell motility is enabled through a variety of ways. These include the activation of phospholipase C, causing the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). This results in 2 products: diacylglycerol (DAG) and inositol triphosphate (IP₃). IP₃ can bind to IP₃ receptors on the endoplasmic reticulum (ER) to cause calcium increase which is needed for cell movement. DAG causes the activation of protein kinase C (PKC) which can phosphorylate transcription factors like NFκB which is the basis for directional movement of the cell. Another mechanism by which cell movement is enabled is by the activation of Rho family of proteins. These proteins facilitate actin-dependent processes which result in pseudopod formation and membrane-ruffling (17).

Where to Start...

I believe it’s always best to start with the basics in order to get a good foundation of knowledge on which to build upon. For this reason, I have gone back to the beginning with regards to chemokines and looked at them as a whole group. The following is what I consider to be the keys points that are relevant to my research.

What are Chemokines?

Chemokines are a type of intracellular signalling molecule that come under the umbrella term of cytokines. They are chemoattractant in nature which means they attract and recruit certain cells and bacteria towards distinct areas in order to carry out a specific function: this is what distinguishes them from other cytokines. They are produced in response to several stimuli including:

·         Bacterial products

·         Viruses

·         Agents that cause physical damage to cells E.g. silica, alum, urate crystals in gout

Due to this fact, it is evident that they are heavily involved in immunity and responding to pathogens. This includes both the innate and adaptive immune systems as they act to induce chemotaxis in the early phases of infection as well as the in the later stages that require leukocyte recruitment (5).

How do they function?

Chemokines act on G-protein coupled receptors (GPCRs) to transmit their signal. These GPCRs have 7 transmembrane helices and are linked to trimeric G-proteins that have an attached GDP protein in the inactive state. The signalling process is by way of the classical GPCR pathway in which activation of the receptor and G-protein causes a dissociation of the three subunits into the α-subunit and the βγ-subunit. These subunits can then go on to activate other intracellular molecules independently and trigger various intracellular pathways including the phospholipase C pathway which can result in an increased intracellular calcium concentration.

For more information visit… http://www.annualreviews.org/doi/pdf/10.1146/annurev.immunol.19.1.397 
 

How are chemokine receptors classified?

There are 4 types of chemokine and correspondingly 4 types of chemokine receptor that are classified based on their amino acid sequence near the terminus of the molecule. These are: 

1.       CC chemokine receptors

These have two adjacent cysteine residues in this region. They are termed CCR1-9 and the genes for these receptors are clustered mostly on chromosome 4. β-chemokines act here.   

2.       CXC chemokines receptors

The cysteine residues on these termini are separated by a single amino acid. CXCR1-6 is used to denote these receptors with α-chemokines acting on them. The genes that encode these receptors are clustered on chromosome 17.

3.       CX3C chemokine receptor

Only one had been discovered: fractalkine or CX3CL1 which binds the sole receptor of this class CX3CR1. It has 3 amino acids between the two cysteine terminal residues.

4.       XC chemokine receptors

The chemokines that bind these receptors are unique in that they only contain two cysteine residues. They attract T cell precursors to the thymus.

 

For this reason, the chemokines that act on these two distinct sets of receptors result in varying physiological effects. The CC chemokine receptors are what I will be elaborating on as I start to look more specifically at the CC chemokine receptor 2 (CCR2) and the diseases it is implicated in.

That’s enough background information! It’s not the most exciting and innovative bit of text but I believe it’s necessary in order to give context to my research. So I hope you haven’t become disheartened as I will endeavour to bring you the latest in chemokine therapeutics and those novel agents that will hopefully make a real difference to sufferers worldwide.