JOINT EPSRC/MRC CHEMICAL BIOLOGY Pharmacological targeting of cytosolic calpain-1: The development of novel inhibitors

Lead Research Organisation: Cardiff University
Department Name: School of Medicine


White blood cells, such as neutrophils and lymphocytes combat infection by leaving the blood circulation to enter the tissues and destroy infecting microbes. However, there are a number of inflammatory disease in which these cells are the cause of the problem. In rheumatoid arthritis, for example, white cells continually leave the blood stream and migrate to the affected joints, where they are inappropriately activated to ?attack? the tissues of the joint. This causes a progressive destruction of the joint.
Unsurprisingly, considerable effort has been made in trying to understand the mechanism by which white cells leave the blood stream so that effective treatments can be devised to stop this. One of the successes has been the use of monoclonal antibodies to block the molecular signals which ?call? white cells to leave the blood stream. However there are problems, with this approach. Firstly, the treatment involves a costly infusion of antibody which must be done in hospital. Secondly, antibodies are highly selectively and can only recognise, and so block, one set of molecules. Since there may be a range of molecules ?calling? to the white cells, any single monoclonal antibody may fail to be effective. We have therefore looked at the mechanics of how the white cells leave the blood stream, because this must be a common event regardless of precisely which calling molecules are involved. We have found that the cells change shape in order to slip through the blood vessel walls. This involves an apparent large increase in the surface area of the cells. We have evidence that this occurs because the membrane of the cells is initially very wrinkled. These wrinkles unfurl when the protein links which hold them together are broken by an enzyme called calpain-1. If this enzyme is inhibited, the unfurling stops and the speed which white cells can leave the blood is greatly reduced. This would be the basis for an anti-inflammatory drug which could be simply taken by mouth. Luckily, we already know of some synthetic molecules which inhibit the enzyme. Furthermore, we know where they bind to the enzyme.
In this project, we plan to use this knowledge to ?tailor? this family of molecules to become more potent and selective only for calpain-1. This will minimise the possibility of unwanted side effects by inhibition of other enzymes which also cut proteins.

Technical Summary

Calpain-1 is a cytosolic cysteine protease which is activated in neutrophils and lymphocytes by a rise in cytosolic free Ca2+ . We have shown that calpain?1 is an important Ca2+ activated enzyme in neutrophils which controls the cell shape change necessary for regulating leukocyte extravasation. However, it is unlikely that pharmacological blanket inhibition of calpains (including calpain-2) would be without unwanted and unacceptable side effects because calpains are expressed in other cell types. In fact, knock out of the common small subunit of calpain (calpain-4) or calpain-2 itself is embryonically lethal. In contrast, calpain-1 knockout mice are viable and apparently healthy.
Although there is accumulating evidence for the signalling role of calpain-1, this has largely depended on pharmacological inhibition. Many of these experimental inhibitors used are ?protease inhibitors? which inhibit cysteine proteases, and do not select between calpain-1 and calpain-2. Clearly, the development of targeted inhibition of calpain-1 will be an asset to the research community and permit the testing of hypotheses relating to the role of calpain-1 both in vitro, and in vivo. There is also an implication for anti-inflammatory therapy. At present, biological agents such as anti-TNF and other cytokines, whilst effective treatment for some inflammatory conditions are both costly and must be administered during a hospital visit by intravenous infusion. Selective calpain-1 inhibitors developed during this project, will have a potent anti-inflammatory effect since they will reduce leukocyte trafficking in response to all cytokines, and hopefully lead to the development of a small molecule inhibitor effective when taken orally.
Most existing ?calpain inhibitors? act on the proteolytic part of the enzyme which is similar to other cysteine proteases. However, alpha-mercaptoacrylate derivatives acts at a Ca2+ binding (regulatory) site of calpain. The binding site and orientation of the inhibitor has been determined from the crystal structure of calcium bound domain VI of calpain at 1.9 ? resolution. It can be argued that since Ca2+ binding and activation distinguishes the calpains from other cysteine proteases, such inhibitors would necessarily have selectivity for calpains. Furthermore, since the difference between calpain-1 and calpain-2 (and others) lies in their different affinities for Ca2+, inhibitors acting at this site may be designed to have selectivity for calpain-1 over calpain-2. The overall aim of the project is therefore to develop alpha-mercaptoacrylate derivatives with high potency and selectivity for inhibition of the cytosolic enzyme calpain-1.
Description MRC Developmental Pathway Funding Scheme (DPFS) - SATRE
Amount £30,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2010 
End 04/2011
Title Novel Calpain inhibitors 
Description Significantly improved inhibitors of cytosolic calpain-1 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact None yet 
Description Small molecule inhibitors of calpain-1 
Organisation MRC-Technology
Department Small Molecules Discovery Unit
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution MRCt are evaluating a novel approach to discovering small molecule which inhibit calpain-1 and would translatable into therapeutic use as anti-inflammatory agents.
Collaborator Contribution Exchange of data and reagents. We have supplied domain VI of calpain and our novel small molecule inhibitors (and fluorescently tagged analogues). Meetings to review progress and discuss direction.
Impact Plan to jointly apply for DPFS funding
Start Year 2010
Title Potential anti-inflammatory therapeutic 
Description Effect calpain inhibitors for potential use in treatment of inflammation 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Actively seeking support
Impact None yet