Review For reprint orders, please contact [email protected]

Calpain-1 inhibitors for selective treatment of rheumatoid arthritis: what is the future? Effective small-molecule treatment of inflammatory diseases remains an unmet need in medicine. Current treatments are either limited in effectiveness or invasive. The latest biologics prevent influx of inflammatory cells to damaged tissue. Calpain-1 is a calcium-activated cysteine protease that plays an important role in neutrophil motility. It is, therefore, a potential target for intervention in inflammatory disease. Many inhibitors of calpains have been developed but most are unselective and so unsuitable for drug use. However, recent series of a-mercaptoacrylate inhibitors target regulatory domains of calpain-1 and are much more specific. These compounds are effective in impairing the cell spreading mechanism of neutrophils in vitro and raise the possibility of treating rheumatoid arthritis with a pill; however, challenges still remain. Improved bioavailability is needed and solution of their precise mode of action should prompt the development of specific calpain-1 screens for novel classes of inhibitors.

Inflammatory diseases such as rheumatoid arthritis (RA) affect a large proportion of the population yet the treatments available are unsatisfactory in a variety of ways; no simple and effective small-molecule therapy has yet been developed. This article will explore a somewhat unheralded target for treatment of RA in just such a way. Calpain-1 is an enzyme that plays a key role in inflammatory response and its inhibition has been shown to alleviate the symptoms of RA in animal models. No inhibitors have yet been identified that are major drug candidates; however, in this article we report on new series of inhibitors that may lead to the holy grail of treating RA with a pill.

David J Miller1, Sarah E Adams1, Maurice B Hallett2 & Rudolf K Allemann*1

RA RA is one of the most common forms of inflammatory arthritis and one of the most prevalent autoimmune diseases [1]. Like all inflammatory diseases, RA is a condition where the body’s natural defense mechanism becomes over stimulated and causes tissue damage. Although the exact causes of this disease are unknown, there are links to both genetics [2] and environmental factors [3]. The damage that occurs through RA causes severe pain and disability to the patient and through development of secondary conditions, such as cardiovascular disease and pulmonary fibrosis, leads to an increased mortality rate [4,5]. In RA, the joints are attacked at the synovial membrane by a number of leukocytes and lymphocytes and the process is initiated via T-cell infiltration [6]. T-helper cells are thought

to initiate the attack; these are activated in response to antigen-presenting cells that are found within the synovium [6]. The presence of these cells causes an influx of macrophages into the synovial lining [7], and, consequently, the release of cytokines is stimulated [6,8,9]. Proinflammatory cytokines such as TNFa and IL-1 act as signaling molecules for the attraction and activation to the area of other leukocytes and lymphocytes, including, neutrophils, chondrocytes and fibroblasts [10]. The activated chondrocytes and fibroblasts secrete metalloproteases and reactive oxygen species that damage the cartilage and tissue within the surrounding area [6]. The constant bombardment of white blood cells over time causes the formation of granular tissue (pannus tissue) that grows over the articular cartilage [5,11]. This granular tissue is composed of synovial fibroblasts and mononuclear cells and leads to further destruction of the cartilage as it contains a high concentration of metalloproteases; this, in turn, exposes the bone, causing more damage to the joint (Figure  1) [11–13]. In addition to the formation of the pannus tissue, synovial fluid within the joint increases in volume and decreases in viscosity, which leads to the loss of lubrication within the joint generating more joint damage (Figure 1). Synovial fibroblasts within the arthritic joint can also spread to other previously unaffected joints [14]. The role of the neutrophil, a polymorphonuclear granulocyte, is often forgotten in RA [15]. Neutrophils are the most abundant form of white blood cells found within the joint, with

10.4155/FMC.13.172 © 2013 Future Science Ltd

Future Med. Chem. (2013) 5(17), 2057–2074

ISSN 1756-8919

School of Chemistry & Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, UK 2 Neutrophil Signalling Group, Institute of Molecular & Experimental Medicine, School of Medicine, Cardiff University, WHRI, Heath Park, Cardiff, CF14 4XN, UK *Author for correspondence: Tel.: +44 29 208 79014 Fax: +44 29 208 74030 E-mail: [email protected] 1

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Review | Miller, Adams, Hallett & Allemann Rheumatoid arthritis

Normal joint

Rheumatic joint

Pannus tissue

Disease-modifying anti-rheumatic drugs such as methotrexate, gold, leflunomide, hydroxychloroquine and sulfasalazine can slow down tissue damage caused by the disease but they also have side effects and their mechanism of action is not fully understood;

n

Biologics such as antibodies to TNF-a (etanercept, infliximab, adalimumab and certolizumab) are the newest form of treatment. These are effective and stop inflammatory cell influx. However, being synthetic antibodies there are problems with their delivery (i.e., by intravenous infusion) and their high costs.

n

Cartilage destruction

Normal

Synovial membrane swelling

Figure 1. Images comparing a normal hand and joint with one afflicted with rheumatoid arthritis. Damage caused to a hand afflicted by (A) rheumatoid arthritis in comparison to (B) a normal hand. (C) A diagram representing a joint with one side afflicted with rheumatoid arthritis and the normal joint.

the highest population being found within the synovial fluid [12,16,17]. Neutrophil cells contain granules that can release their toxic contents into the surrounding area and, therefore, can cause severe damage to the cartilage and bone [15,18,19]. Inhibition of inflammatory cell migration from the blood into inflamed tissue would have a major therapeutic benefit for all inflammatory diseases. As RA is the most widespread and important of the inflammatory diseases, the current treatments for this will be considered. However, similar treatments would apply to other inflammatory diseases. Key Terms Rheumatoid arthritis:

Common inflammatory disease affecting approximately 1% of the wordwide population.

Calpain: A group of cysteine

proteases involved in the migration of white blood cells to damaged tissue.

Inflammatory diseases:

Common ailments in which the body’s defense systems cause damage to the patient.

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Current treatments & their problems At present there are four treatment options for RA: n Non-steroidal anti-inflammatory drugs such as ibuprofen and COX-2 inhibitors. These are used to relieve pain and stiffness but they do not reverse or slow down the progression of tissue damage; Corticosteroids also reduce pain and can reduce inflammation and inflammatory cell influx. However, they can only be used on a short-term basis because of the side effects associated with long-term corticosteroid usage;

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Small-molecule intervention in inflammatory cell influx is an attractive therapeutic alternative to the above treatments. Effective inhibition of inflammatory cell influx to inflamed joints and other tissues can be achieved by inhibition of enzymes involved in the chemotaxis of inflammatory cells. As activation of calpain-1 within immune cells (especially neutrophils and lymphocytes, vide infra) is required for the cells to undergo rapid shape change as they spread on to the endothelium and extravasate into the extravascular tissues, reducing this would control leukocyte trafficking and reduce the progress of destructive inflammation [20,21]. It would be significantly less costly than the biologics and likely to have fewer side effects. Neutrophils Neutrophils are a form of granular leukocyte and are the most abundant group in the immune system making up 40–65% of all white blood cells. They are typically the first cells to reach the site of an infection or trauma [22]. Granular leukocytes are so called as they contain cytoplasmic granules, the contents of which are highly toxic towards foreign bodies [22]. These granules contain a wide variety of proteinases and other hydrolases as well as proteins that have the capability to generate reactive oxygen species [19]. Upon normal activation at the site of action, the cells are phagocytic and encapsulate foreign bodies in a vacuole [20]. The cell then releases the contents of the granules into the vacuole to destroy the contents [19,23,24]. Neutrophils have a relatively short lifetime of 5–7 days within the body [22]. Once the cell has reached the end of its lifetime it undergoes apoptosis and the remnants of the cell are taken up by macrophages [24]. For these cells to reach their target site of infection or inf lammation, small-molecule chemoattractants such as N-formyl peptides future science group

Calpain-1 inhibitors for selective treatment of rheumaoid arthritis: what is the future? and IL-8, are released through the initial damage that is caused in the local area [22,25,26]. Through these chemoattractants the cells are triggered to migrate towards the site and stimulate the activation of integrins – cell adhesion molecules [26]. To do this, the neutrophils undergo a rapid shape change to adhere to the endothelium, since they are roughly spherical in shape within the bloodstream (F igure  2) [20,27]. In this spherical shape, the cell surface membrane has numerous wrinkles all along the surface, which are held together through two different protein scaffolds [20]. Membrane-bound proteins, such as L -selectin and b2-integrin, are localized upon the peaks and valleys of the wrinkles along the cell membrane of the neutrophil, respectively [27]. The glycoprotein L -selectin has been proposed to bind to the intracellular protein actin through a protein ‘bridge’ ezrin, while actin is linked to the membrane-bound protein b2 -integrin through talin (Figure 2) [20,28]. In response to the chemo­attractants, Ca 2+ ions are released, leading to the activation of the cystolic cysteine protease calpain-1 at the plasma membrane [21,29]. The activated protease then cleaves the protein bridges, talin and ezrin holding the wrinkles in place, which allows the cell membrane to spread and adhere to the endothelial membrane of the blood vessel (Figure 2) [20,30]. The membrane-bound proteins are a vital component in the cell’s ability to adhere to the endothelial membrane. Once activated, the cell goes through a rolling motion, where the L -selectins and b2-integrins adhere to the endothelial wall [22]. In order for the chemotaxis to occur, the cell utilizes the actin filaments below the cell membrane, as protrusions push the membrane forward [22,31]. For the cell to detach at the rear calcium and calcineurin are required for the recycling of b-integrins [32]. The cell then migrates towards an ever increasing concentration of chemoattractants until the site of damage is reached [33]. The calpain family The calpain family is a group of calciumactivated cysteine proteases within the papain superfamily [34]. There are 15 known isoforms in the calpain family, some of which are ubiquitously expressed throughout the human body, such as the two most studied members calpain-1 and calpain-2 [35], while others such as calpain-3 (p94), which is found in the skeletal muscle, are tissue specific [36]. These enzymes future science group

| Review

Selectin Ezrin Actin Calpain-1 activation

β2-integrin Calpain-1 activation

Talin Actin

Figure 2. Neutrophil in the bloodstream with wrinkles on the cell surface membrane. Detailed structures of the individual wrinkles with L-selectin/ b2-integrin bound to ezrin/talin, which is bound to the intracellular protein actin (peak of the wrinkle/’valley’ of the wrinkle).

are modulator proteases meaning that instead of hydrolyzing a protein substrate in order to destroy it, such as proteasomes and caspases, the enzyme hydrolyses substrates to modify and repurpose them within the body [37]. Inhibition of calpain-1 has been shown to impair TNF-a mediated neutrophil adhesion to fibrinogencoated surfaces [38], reduce inflammation in rats [39] and ameliorate the symptoms of RA in mice [40]. It has also been reported to be an activator of NFkB – transcription factor with a key role in inflammation [41]. Clearly it is, therefore, a valuable potential target for clinical intervention in the treatment of this disease. In contrast, inhibition of calpain-2 has been found to be embryonically lethal [42] and so in targeting calpain-1 specifically for the treatment of RA, it is essential to understand the structure, mode of action and regulation of both enzymes. www.future-science.com

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Review | Miller, Adams, Hallett & Allemann „„Calpain-1

& -2 Calpain-1 and calpain-2 (with associated gene names of CAPN1 and CAPN2, respectively) are heterodimeric calpains composed of a large subunit known as CAPN1 and CAPN2 in the two isoforms respectively, and a small subunit that is known as CAPNS1 [43]. The large subunit comprises four domains and the small subunit two (Figure 3) [44]. The N-terminal domain of the large subunit is the so-called anchor a-helix, which is followed by the CysPc domain, the proteolytic core of the enzyme [38,45]. The third domain within the large subunit is the C2L domain that is similar to C2 domains that are phospholipid binding domains found in phospholipases [38,45,46]. The fourth domain within the large subunit is the penta-EF hand domain (PEF(L))-calcium-binding domain, this comprises five EF hand motifs [38,45]. The two domains that are found in the small subunit (CAPNS1) are the N-terminal glycine-rich (GR) domain and the PEF(S) calcium binding domain, which is also composed of five EF hand motifs [38,45]. CAPN1 comprises 714 amino acids [47] and is 14 residues larger than CAPN2 [48]. The

Key Term Calapastatin: Endogenous inhibitor of calpains 1 and 2.

„„Domain

CysPc PC1

PC2

Anchor helix

C2L

PEF(L)

GR

PEF(S)

CAPN1/CAPN2

CAPNS1

C2L PEF(L)

sequence similarity between the large subunits of these two isoforms of calpain is 62% [48]. CAPNS1, also known as the regulatory subunit, is composed of 266 amino acids and is identical in both calpain-1 and calpain-2 [49]. The most significant apparent functional difference between calpain-1 and calpain-2 is the concentration of calcium that is required to activate them [44]. Approximately 50 µM calcium is required for the activation of calpain-1 and approximately 0.35 mM for the activation of calpain-2. They are also named µ-calpain and m-calpain, respectively [44]. The concentration of calcium ions that is required to activate these two isoforms in vitro is much higher than the levels of calcium ions that are found in vivo [50]. Hence there must be other factors involved in the activation of these proteases within the body. One of these is the association of other molecules to the protease, such as is the case with phosphorylation, which lowers the concentration of calcium ions required for activation [50]. Both isoforms of calpain are inhibited by the same endogenous and presumed regulatory inhibitor, calpastatin, a highly specific inhibitor for conventional calpains [44].

Anchor helix

structure Both calpain-1 and calpain-2 contain similar domain structures comprising a large subunit and a small subunit, the latter of which is identical between the two forms. There is only a hybrid crystal structure of calpain-1 currently available, which contains the PEF(L) domain and the anchor helix of calpain-2 [51]. Consequently, all structural analyses are of individual domains of calpain-2. However, the hybrid structure of calpain-1 revealed that the domains of calpain-1 are structurally similar to those of calpain-2 [51–53]. N-terminal anchor a-helix The anchor a-helix domain is a small domain, in CAPN2 it is 19 residues in length (Figure 4), and leads into the CysPc domain [52]. Sequence alignment of CAPN1 with CAPN2 indicates that this anchor a-helix is ten residues longer than the a-helix in CAPN2 [54]. The increase in the length of this domain, in calpain-1, has been shown to account for approximately 10% of the difference in the concentrations of calcium that is required for activation [55]. When the protein is inactive the a-helix interacts with the PEF(S) domain (Figure 4) this enables clamping of the two domains together [52]. „„The

CysPc PEF(S) GR

Figure 3. Overall structure of calpain-1 and -2. (A) Domains of both CAPN1 and CAPN2 along with the CAPNS1. (B) The structure of inactive calpain-2 (PDB:1KFU). Image rendered in PyMOL, as are all subsequent structural images [201].

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future science group

Calpain-1 inhibitors for selective treatment of rheumaoid arthritis: what is the future? Upon activation the most common theory for the fate of this domain is autolysis since this enables activation with a lower concentration of calcium ions in calpain-2 in vitro [56]. However this autolysis also causes the heterodimer to become unstable and dissociation occurs, which in turn may lead to a loss of substrate specificity for calpain-2 [57]. CysPc proteolytic domain The CysPc proteolytic domain contains the catalytic dyad of Cys and His. It is composed of two subdomains that are known as PC1 and PC2 [52]. PC1 contains the catalytically active Cys residue, while the active site His is in PC2 [52,53]. PC1 and PC2 are found in calpain-1 and calpain-2. The secondary structure of the PC1 subdomain is composed of two antiparallel b-sheets and a series of ahelices [53]. The active site Cys residues are found at positions 115 and 105 for calpain-1 and-2 [53]. PC2 is also composed of two antiparallel b-sheets and a series of a-helices, this subdomain completes the catalytic dyad with the His [53]. The residues of the catalytic dyad reside on the interface between the two domains, but are held apart through structural restraints to prevent hydrolysis of substrates until activation. Cys and His are held 8.17 Å away from one another in the inactive form of calpain-2 (Figure 5) [52]. One of these structural restraints is the anchoring helix bound to the PEF(S) domain, where the binding of calcium ions to the PEF(S) domain causes structural movement to release the anchor peptide allowing for the two subdomains of the proteolytic core to move towards one another [53]. Another structural restraint is provided by the acidic loop of the C2L domain that interacts with a series of basic residues in the PC2 subdomain and acts like an electrostatic switch upon binding of calcium (Figure 6) [58]. This domain binds two calcium ions and this contributes to the movement of the separated portions of the catalytic dyad towards one another to form the active enzyme [59].

| Review

CysPc C2L

„„The

C2L domain The C2L domain is a 130-residue domain that acts as a bridge between the CysPc domain and PEF(L). This domain folds into a pair of four stranded parallel b-sheets and is broadly similar to a C2-binding domain [53]. C2 binding domains are known to bind phospholipids but in order for this to occur normally there is an initial step, whereby most C2 domains bind calcium [60]. An acidic loop lies within this domain

PEF(S)

PEF(L)

Figure 4. The N-terminal a-helical domain of calpain-2 and its position in the overall structure. (A) Highlights the 19-residue N-terminal a-helical domain of calpain-2 (black). (B) The interaction between the N-terminal a-helix and the PEF(S) domain that, among other interactions, holds the catalytic active site open, individual side chains mostly involved in hydrophobic interactions are shown as sticks.

CysPc

8.17 Å

„„ The

future science group

Figure 5. The CysPc domain of calpain-2 and the catalytic dyad within it. (A) A cartoon representation of the CysPc domain of calpain-2 and its position within the protease. (B) The catalytic dyad in the inactive conformation within the CysPc domain of calpain-2, with the distance between C105 and H286 at 8.17 Å.

www.future-science.com

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Review | Miller, Adams, Hallett & Allemann (Figure 6).

When the whole of the 130-residue domain is expressed separately from the rest of the enzyme, it binds calcium with an affinity similar to PEF(L). The concentration of calcium ions required to bind the C2L domain is dramatically reduced when phospholipids are also bound to the domain [61]. The phospholipid binding nature of this domain gives evidence of how the protease is capable of migrating in its inactive form from the cytosol to the site of action at the membrane where activation of the enzyme occurs [62]. This series of acidic residues also have an electrostatic interaction with basic residues that are found in the PC2 subdomain (Figure 6). These hold the cleft between PC1 and PC2 open and therefore keep the protease in an inactive conformation [58]. As well as the acidic loop there are other acidic residues in calpain-2 such as Glu504, that interact with the PC2 subdomain; this resides on the linker between the C2L domain and the PEF(L) domain [63]. Mutations to this acidic loop can cause lowering of the concentration of calcium that is required for activation in vitro, demonstrating that these electrostatic interactions are integral for keeping the protease in its inactive

CysPc

Acidic loop

Asp Lys Glu Lys

form [63]. Upon activation, disruption of the electrostatic interactions occurs and this allows the conformational change to active protease to occur. „„PEF(L)

& PEF(S) domains The PEF(L) calcium binding domain differs between the two isoforms of calpain. As noted above, the concentration of calcium required to activate calpain-1 is lower than that required for the activation of calpain-2 [44]. As there is no full crystal structure of calpain-1 and therefore there is no structure of its PEF(L), any secondary and, for that matter, tertiary structure that could be used to determine the reason why there is a discrepancy in binding that cannot be confirmed at this point. It has been noted that the calcium binding residues in the EF-hand loops are highly similar, therefore, the reason for the discrepancy in the concentration of calcium required for activation cannot be based upon this [64]. Transformation of the PEF(L) residues of calpain-1 into the residues found in calpain-2 and vice versa demonstrated that these domains are integral for the concentrations of calcium required for activation of the two isoforms [55]. The overall sequence similarity between the PEF(L) domains found in calpain-1 and calpain-2 is 48% in the human versions of the proteases [49]. The PEF(L) domain of calpain-2 comprises eight a-helices that form five individual EFhands (F igure  3). Four of these loops bind calcium ions and the fifth coordinates to the PEF(S) domain through a so-called EF-handshake. PEF(S) is also composed of five EFhands, four of which bind calcium and the fifth binds to its corresponding EF-hand in PEF(L). This domain are identical in both calpain-1 and -2 [44]. The regulatory subunit is so-called due to the fact that it is a layer of protection in order to prevent the over-activation of the protease. PEF(S) and PEF(L) have very similar structures [65]. A crystal structure with calcium bound to this domain indicated very little movement of the EF-hands [65]. The minimal movement of these domains suggests that these two calcium binding domains play a limited role in activation of the protease for catalysis [66]. „„The

Figure 6. The C2L domain of calpain-2 and the acidic loop within its structure. (A) A cartoon representation of calpain-2 C2L domain, in black and (B) close up of the acidic loop (residues 392 to 402, represented as sticks) and its electrostatic interactions with the basic residues in the CysPc domain (light gray) that keep the protease in an inactive conformation.

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GR domain The GR domain comprises 95 residues with 40 being glycine [45]. There is no resolved crystal structure of this domain due to its flexible nature [52]. In addition to the glycine within this domain, there are also five proline residues that possibly form a section of rigidity [44]. The future science group

Calpain-1 inhibitors for selective treatment of rheumaoid arthritis: what is the future? flexibility of the domain implies that it may be used to bind the heterodimeric protein to other molecules or structures such as the cell membrane [44]. Calpastatin The endogenous 70 kDa inhibitor of calpain-1 and calpain-2 consists of five domains [66, 67]. It has no sequence similarity to any other known protein [68,69]. The first domain is the N-terminal L-domain and the other four domains are individual inhibitory domains, each of which binds a single molecule of the heterodimeric protease [67,68]. The domains are known as the L-domain (N-terminal) and domains I–IV for each of the inhibitory domains (Figure 7) [70]. Each of the inhibitory domains has differing potency when binding a molecule of calpain-1 or calpain-2. The most potent domain of calpastatin is domain I (Kd = 4.5 pM), followed by domain IV (Kd = 50 pM), which is more potent than domain III (Kd = 0.6 nM) and domain II (Kd = 4.0 nM) [71]. The inhibitory domains are composed of approximately 140 amino acids each and contain three inhibitory regions that bind to different areas of the heterodimeric structures of the proteases [68]. The inhibitory regions are known as regions A, B and C (Figure  7). Solution of a calcium-bound co-crystal structure of calpain-2 and inhibitory domain IV of calpastatin showed that inhibitory regions A and C are both a-helical and reside within hydrophobic grooves of PEF(L) and PEF(S), respectively [72]. Inhibitory region B is inherently unstructured and stretches to either side of the active site cleft. The N-terminal end of region B interacts with the C2L domain [72]. Peptide inhibitors that are based on the sequence of inhibitory region B were found to be capable of inhibition of calpain-1 and calpain-2 [73]. Interestingly, in the absence of inhibitory region B, regions A and C act as activators of the cysteine protease [74]. Activation of calpain-1 & -2 Activation of calpain-1 and calpain-2 occurs through binding of calcium ions, which, in turn, leads to conformational changes that enable the enzyme to reach its active form. Calpain-2 coordinated to one domain of calpastatin is the only known calcium-bound crystal structure [63]. Hence, a variety of alternative techniques have been used to investigate indirectly the conformational changes that occur during calciummediated activation. These include mutation of residues within the enzyme [64] and the solution future science group

L

I

II

III

IV

A B C

A B C

A B C

A B C

| Review

Calpastatin

Inhibitory region C Inhibitory region B

Inhibitory region A Inhibitory region B

Figure 7. The domain structure of calpastatin and its interactions with calpain-2. (A) A schematic delineating the inhibitory domains of calpastatin; domain L and domains I–IV, the inhibitory regions, A, B and C are highlighted by a pale gray box and regions of calpastatin not bound to calpain when complexed are shown in black. (B) A cartoon representation of inhibitory regions A, B and C (labeled) bound to the heterodimeric structure of calpain-2; the inhibitory region B is mostly an unstructured chain traversing the length of the C2L and CysPc domains of calpain (PDB:3BOW).

of the structure of isolated calcium-binding domains [61]. The penta-EF hand domains, PEF(L) and PEF(S), show very little movement when calcium binds and so are thought to have more of a regulatory role rather than taking major roles in the activation of calpain [66,75]. Both of these domains are important, however, for the determination of the overall concentration of calcium required in the activation process [55,76]. The largest calciuminduced motion observed occurs in the N-terminal EF-hand motif (EFH-1) of PEF(S) and this implies that a similar motion occurs with EFH-1 of PEF(L) upon calcium binding. This is joined to the C2L domain through a long linker [66] that extends and disrupts some of the electrostatic interactions between the C2L domain and the PC2 subdomain [77,78]. The interactions between PC2 and C2L play a vital role in constraining the protease in the inactive form [58]. Mutations at the interfaces of these domains dramatically decrease the concentration of calcium required for www.future-science.com

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Review | Miller, Adams, Hallett & Allemann

6.20

3.54

Figure 8. Details of the active site of calpain and its movement upon calcium binding. (A) Inactive CysPc domain of calpain-1, derived from a hybrid structure of calpain-1 and calpain-2 (pale gray), overlaid with the calcium bound CysPc domain which is in the active conformation (darker gray). A close up of the catalytic dyad, (B) the inactive protease and (C) the active protease.

activation [45]. Glu504 and Lys234, for example, are especially significant as they form an important salt bridge interaction that is disrupted upon alteration of either residue [64]. The binding of calcium to the PEF(S) domain at the second EFhand motif from the N-terminus is presumed CysPc

Anchor helix

PEF(S) C2L PEF(L)

Figure 9. Postulated two-step activation of calpain. Calcium initially binds to the PEF domains releasing the electrostatic interactions that hold PC1 and PC2 apart. Calcium then binds to PC1 and PC2 causing an alignment of the proteolytic core.

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to cause repulsive interactions with the anchor helix so that there is a release in the constraints that hold the active site subdomains apart. In the absence of any structural evidence but due to its similarity to C2 domains that bind calcium in the presence of phospholipids, the C2L domain is assumed to bind calcium also [60,61]. The disruption of the electrostatic interactions between the proteolytic core and the auxiliary domains allows PC1 and PC2 to align with one another but in order to complete the activation the two subdomains also require calcium [79]. Crystallization of the proteolytic core of calpain-1 alone revealed that the two subdomains contain a calcium-binding site each (Figure 8) [59]. The proteolytic core is in itself a calcium-activated cysteine protease with a calcium ion requirement of 40 µM relative to the 50 µM of calcium that is required for full length calpain-1 Hence a two-step activation route can be postulated for the activation of calpains-1 and -2. The first step requires a release of the restraints that hold the proteolytic core in an inactive conformation. This occurs through the binding of calcium to the PEF domains on both subunits. Once calcium is bound, the electrostatic interactions between the C2L domain and the CysPc domain are disrupted, as well as the interactions between the anchor helix and PEF(S). Following this, calcium binds to the proteolytic core allowing PC1 and PC2 to rotate towards one another and form the catalytic dyad (Figure 9). Factors that lead to activation in vivo The typical concentration of calcium within the cell is 1 µM. Locally high concentrations may activate calpain during calcium influx [45], such as just beneath the membrane in neutrophils where the concentration can reach up to approximately 30 µM, but this remains too low for activation of either calpain isoform [29]. Other factors must therefore contribute to calpain activation [80]. These include the binding of phospholipids,[81,82] autolysis, the binding of activator proteins [83–85] and phosphorylation [51]. Phospholipids have been observed to bind to the C2L and GR domains of calpains-1 and -2 [61,67]. This decreases the concentration of calcium that is then required for activation implying that this process may also increase calcium sensitivity in vivo [81,82]. This is consistent with studies that show that calpain is mainly active at the cell membrane [86,87]. Phospholipid future science group

Calpain-1 inhibitors for selective treatment of rheumaoid arthritis: what is the future? binding has also been found to lower the calcium requirement for autolysis of the N-terminal a-helix [88]. This autolysis in turn allows for a further lowering of the calcium requirement for activation in vitro and so presumably in vivo (Figure 10) [81,88–90]. Activator proteins may also contribute to increased calpain sensitivity toward calcium. Several proteins have been observed to associate with calpains-1 and -2 and lower the calcium requirement for activation. These include acetyl-CoA binding protein [83], UK114, a tumor antigen [84] and a protein of otherwise unknown function found in human neutrophils [85]. function & known substrates Definitive physiological roles for calpain-1 and -2 in vivo have been difficult to determine because gene knockouts are often lethal and known inhibitors lack selectivity [91]. Many substrates of calpain-1 and calpain-2 have been found in vitro and used to predict potential functions of the proteases. The two isoforms have almost identical substrate specificity with over 100 substrates discovered to date [91]. These include a variety of cytoskeletal proteins, several kinases and phosphatases, some membrane-bound proteins and several cytosolic proteins [44,91,92]. Cytoskeletal protein substrates include fodrin [93], ezrin [94] and talin [95]. Many calmodulin-dependent proteins either membrane bound or in the cytosol are susceptible to calpain mediated hydrolysis [92]. These include membrane-bound enzymes such as Ca 2+-ATPase [96] or enzymes that reside within the cytosol during apoptosis, for example caspase 12 and calcineurin [97,98]. Through sequence ana­lysis the most common amino acids to occur at position P1 and P1´, either side of the cleavage site are tyrosine, lysine or arginine at P1 and serine at P1´ (Table 1) [99]. However, a screen using a synthetic peptide library found that the optimum amino acids were either a leucine or a phenylalanine at P1 and a methionine, alanine or an arginine at P1´ (Table 1), although this ‘optimum’ sequence has yet to be identified within a eukaryotic protein [100]. Proline residues are often present at certain positions within the sequence of the substrate suggesting that the cleavage site is unstructured and that a lack of ordered secondary structure may be a requirement for the recognition of the substrate by the protease [99]. There is a notable lack of correlation between the ‘optimum’ sequences found from substrates in vitro and the peptide library screen however

| Review Active

Inactive

Ca2+

Ca2+

„„Physiological

future science group

Figure 10. A proposed activation mechanism of calpain via membrane association (phospholipid binding) and subsequent autolysis. The inactive protease binds to the cell membrane and then autolysis of the anchor helix occurs. This lowers the overall calcium concentration required for protease activation.

highlighting the difficulties associated with identifying definitive substrate specificity and in vivo functions of calpains-1 and -2. Through the specific sequences of amino acids that are required for substrate activity, numerous susceptible proteins have been identified and their nature can imply roles for the protease in vivo. Caution must be used in the ana­lysis however since these substrates have been identified in vitro where the environment differs greatly from that within a cell. Postulated roles for the calpain isoforms include involvement in signal transduction [101], the cell cycle [91], platelet function [102], erythrocyte deformability [103], apoptosis [104]and cell spreading and migration [105]. It is this final role for calpain, and specifically calpain-1, that has made it a target for intervention in inflammatory diseases such as RA. From the apparent roles they play in vivo it becomes clear that distinguishing calpain-1 from calpain-2 with smallmolecule inhibitors is crucial since disruption of the activity of calpain-2 can be disastrous [42]. Table 1. The preferred amino acids at the cleavage site for calpain-1. Sequence P3 A

†

B‡ † ‡

P2

P1

Trp > Pro

P1´

P2´

Leu > Thr Lys > Tyr Ser Pro > Val > Arg Phe > Leu Leu > Val Leu = Phe Met > Ala > Glu > Pro Arg

P3´ Pro > Trp Arg > Lys

Derived from the sequences of 49 known substrates. Derived from a screen of a synthetic peptide library.

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Review | Miller, Adams, Hallett & Allemann Cell cycle Both isoforms have been shown to affect progression through the cell cycle [106]. Microinjections of calpain-2 at different phases of the cell cycle caused rapid progression to the next phase, including acceleration of mitosis and transitions from the metaphase to the anaphase and from the prophase to the interphase [107]. Calpain-1 inhibition in fibroblasts has been shown to stop the growth of these cells at G1 and prevent progression to S phase [108]. Platelet function Both isoforms have been found within platelets although there are conflicting reports over which is dominant [109,110]. The inhibition of calpain in platelets demonstrated an essential role in the secretion, aggregation and the spreading of platelets [111]. Apoptosis Programmed cell death involves both calpain-1 and calpain-2. In the earlier stages of apoptosis calpain-1 is activated through the release of calcium from the endoplasmic reticulum [112]. The activated protease then hydrolyses the proapoptotic protein Bid, which in truncated form associates with other proapoptotic proteins [113]. Calpains-1 and -2 are also linked to the cleavage and activation of caspases during apoptosis [97]. Furthermore, they have been found within mitochondria and associated with the latter stages of apoptosis, through the truncation and transportation of apoptosis inducing factor from the mitochondria [114–116]. Cell spreading & migration Calpain-1 has been shown to play an integral role in the ability of cell membranes to spread and hence enable the migration of neutrophils (vide supra) [20]. Both isoforms may also be important for the motility of other cell types [117,118]. Many known in vitro substrates such as ezrin [119], talin [120] and b2-integrins [121,122] form focal adhesion complexes that span the plasma membrane and protrude out into the extracellular space [123]. These complexes enable adhesion of the cell to the endothelial wall to begin the process of extravasation. Further to this, calpains are also implicated in performing similar roles in T cells and fibroblasts. Calpain-2 is a vital component in the ability of T cells to form integrin interaction complexes and hence in the cell spreading process [118]. Inhibition of calpain-2 through the 2066

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over-expression of calpastatin within fibroblasts prevented the ability of the cells to form protrusions within the cell membrane, which in turn prevented migration of the cells [124,125]. Since most of the experimental evidence for this has been obtained with inhibitors however, conclusions must be taken with care due to the poor specificity of most inhibitors for individual calpain isoforms. The various roles of calpains in diseases have been reviewed recently [126–130] and so will not be covered here. However, despite the observation of the accumulation of neutrophils in the synovial fluid of patients with rheumatoid arthritis little attention has been paid to their role in this disease and the part played by calpain-1 [131]. Inhibition of calpain The optimum selective inhibitor for both calpain-1 and calpain-2 is the endogenous inhibitor calpastatin. The main problems associated with its use for in vivo experiments are that, due to its size it is not cell permeable, and that it does not discriminate between the two isoforms [42]. Efforts have therefore been made to develop potent, cell-permeable inhibitors that are selective between the isoforms. Calpain inhibitors have recently been reviewed extensively so only a brief overview will be presented here [132,133]. Calpastatin-based inhibitors A variety of inhibitors have been developed around the sequence of the inhibitory region B of calpastatin [133]. The peptide CPB-1 is an example and has similar properties to the endogenous inhibitor. It was found to be a potent inhibitor of calpain with a K i of approximately 0.2 nM but it did not penetrate the cell membrane [73]. Variants of CPB-1 have been developed that can cross the cell membrane and are highly selective for calpains (>1000-fold over cathepsin-L) but as with calpastatin, they do not discriminate between the isoforms [134]. „„Inhibitors

containing warheads Inhibitors containing warheads react with the active site cysteine [132,135]. E-64 (1) contains an epoxide-based warhead and is known to irreversibly react with the active site cysteine. The peptide is derived from a bacterial protein and is nonselective with respect to calpain-1 and -2 and inactivates a wide range of cysteine proteases [136]. A derivative of this inhibitor has been developed, WRH(R,R) (2; Figure 11)that has greater future science group

Calpain-1 inhibitors for selective treatment of rheumaoid arthritis: what is the future?

| Review

N HN NH H2N

O

H N

N H 1

O

N H

O

O

H2N OH

O

HN

N H

O

O O

O

HN

2

NH2

HN

O

O

H N

Figure 11. Peptide-based warhead inhibitors E-64 (1) and WRH (R,R) (2) .

selectivity towards calpains over other cysteine proteases, such as cathepsins. WRH(R,R) has IC50values of 0.46 µM and 0.10 µM against calpain-1 and calpain-2, respectively (IC50 > 10 µM against cathepsins B, L and K) [137]. Leupeptin (3) is a peptide that contains an aldehyde and also targets the catalytic cysteine residue. It was sourced from various species of Streptomyces and other Actinomycetes and is highly potent towards calpain-1 and calpain-2 with K i values of 0.27 µM and 0.38 µM, respectively (Figure 12) [132,138]. Leupeptin is also a general cysteine protease inhibitor and inactivates several serine proteases with greater potency than calpains, moreover it is also not cell permeable and so an unattractive inhibitor for calpains for use in biomedical application [139]. Calpeptin (4) is an example of a leupeptin analog that can cross cell membranes and inhibits calpain-1 and calpain-2 with IC50 values of 0.010 µM and 0.014 µM, respectively [140]. ALLN (5) and ALLM (6) are two widely used aldehyde based inhibitors. Both compounds are potent cell permeable inhibitors of calpain-1 and -2 [132,141]. However, they are more potent inhibitors of certain cathepsins than calpain (e.g., K i of ALLN for calpains-1 and -2 ~200 nM vs 0.5 nM for cathepsin L) [142]. without warheads Ideally, calpain-1 inhibitors, which can be used as drugs, must be reversible inhibitors and not inhibit calpain-2. Non-warhead-based inhibitors have been discovered from natural sources as well as through synthesis. With an IC50 of 87 pM for calpain-1, the synthetic, peptidebased compound 7 is the most potent known inhibitor to date (Figure 13) [143]. This inhibitor does not interact with other cysteine proteases such as papain or cathepsins, although no data have been reported for inhibition of

calpain-2  [143]. It contains a biphenyl linker between two peptide chains and is thought that it prevents calpain activation through chelation of calcium ions [143]. Non-peptidic calpain inhibitors include penicillide 8 , a polyketide that was isolated from Penicillium species. It has activity against calpain-2 with an IC50 of 7.1 µM and did not inactivate papain at a concentration of 200 µM [132,144]. Quinoline derivatives have been shown to have inhibitory properties towards calpain-1, with a derivative of 3-quinolinecarboxamide (9) displaying an IC50 of 0.5 µM compared with IC50 values of 25 and 22 µM versus cathepsins B and L, respectively [145]. Another quinoline derivative (10) has inhibitory properties towards calpain-1 with an IC50 of 0.28 µM. No inhibition (IC50  200 µM) of cathepsin B, papain, trypsin and thermolysin, although they display modest potency against the calcium-modulated activity of calcineurin (K i = 84 µM) [148]. These compounds are widely used and commercially available calpain inhibitors. They were prepared originally as inhibitors of calpain-2 in a study aimed at limiting ischemic stroke damage [148], but this work inspired a more recent investigation into their use in the prevention of neutrophil cell spreading and hence as a possible treatment for inflammatory disease. In this work a series of analogs of 11 and 12 were prepared to investigate the structure– activity relationship regarding the position and nature of the halogen substituent on the aromatic rings. This resulted in the synthesis of bromoindole a-mercaptoacrylates, such as 13, which, to date, are the most potent allosteric inhibitors of calpain-1 (K i = 2–7 nM). They are able to penetrate the cell membrane and significantly impair the spreading of live neutrophils (Figure 15) [149]. Future perspective Calpains-1 and -2 are calcium-ion-dependent cysteine proteases that are intimately involved in several cellular processes and have been targeted for drug development. Since calpain-1 is crucial in the chemotaxis of inflammatory cells towards sites of damage and is an activator of NFkB it represents a highly promising candidate future science group

Calpain-1 inhibitors for selective treatment of rheumaoid arthritis: what is the future?

| Review

210

Area (µM2)

190 170 150 130 110 90 70

0

1

Time (min)

2

3

Figure 15. Interaction of calpain domain VI with a mercaptoacrylate inhibitor and the suppression of neutrophil cell spreading behavior. (A) A cartoon representation of PD150606 (11) bound to the same region of porcine PEF(S) (PDB:1NX3) as calpastatin region C, the inhibitor is represented as sticks. (B) Inhibition of neutrophil spreading by indole mercaptoacrylate calpain-1 inhibitors. The graph shows the area of neutrophils immediately in contact with a microscope slide glass surface and the change over the subsequent 3 min. The upper images are for cells incubated with DMSO (0.1%) and the lower curves are for cells incubated with inhibitors having the following halides at position 6 on the indole ring of the mercapotacrylate inhibitor (top to bottom) F, Cl and Br (10 µM).

for small-molecule intervention in the treatment of diseases such as RA. The structure and mechanism by which these enzymes are activated and carry out their chemistry is becoming increasingly clear. This is particularly true of calpain-2 but since calpain-1 is the target for RA treatment there is a need for more structural biology work on this isoform. Many of the inhibitors discovered for these enzymes have aided the analyses of their structure, function and activation but cannot discriminate between the isoforms and are not sufficiently selective to be useful as drug candidates. The recent discovery of a-mercapotacrylates that are cell permeable, reversible and selective calpain inhibitors and prevent morphology changes in live neutrophils is an exciting development in the inhibition of these enzymes. Several challenges remain before R A can be treated successfully by oral administration. Mercaptoacrylate inhibitors are not likely sufficiently bioavailable to be good drug candidates and, due to the presence of free sulfhydryls, they are prone to oxidation. They also do not possess sufficient water solubility for efficient transport to the site of action (there are no data available on their pharmacokinetics). Their mechanism of action against the calpains is also not yet clear. Although they bind to the PEF(S) domain of calpain, this is a domain shared by the two isoforms of the enzyme and unlikely to be the source of how they discriminate between them. With its similar structure future science group

and sequence similarity, it is possible that they also bind to the PEF(L) domain that varies from calpain-1 to calpain-2. No information is yet available on how they bind to this domain. Such detail will aid in the design of further generations of inhibitors that possess improvements in selectivity, bioavailability and potency. Mercaptoacrylate compounds selective for calpain-1 may also be tagged with fluorescent chromophores and prove useful in the development of calpain-1-specific screens to discover other classes of calpain-1 specific inhibitors. Analysis of the inhibitory activity of compounds 7–10 and their analogs against both calpain-1 and calpain-2 to unearth any unknown specificity would also be useful. Bearing all this in mind, with the discovery of potent, selective, calpain-1 inhibitors already in hand, the future looks bright for calpain-1 as a target in the treatment of RA and other inflammatory diseases. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert t­estimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. www.future-science.com

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Review | Miller, Adams, Hallett & Allemann Executive summary Rheumatoid arthritis Rheumatoid arthritis, a common inflammatory disease, may be treated through limiting the influx of damaging white blood cells into otherwise healthy tissue. Current treatments & their problems „„

Current treatments for rheumatoid arthritis include use of non-steroidal anti-inflammatory drugs, corticosteroids and anti-TNF-a antibodies such as adalimumab. These are either limited in effectiveness, expensive or invasive. Neutrophils „„

The most common form of white blood cell found in damaged joints. Chemoattractants induce them to migrate to sites of damage and they move from the blood stream initially through release of membrane restraints that allow them to change shape and extravasate through the blood vessel wall. The calpain family „„

A series of Ca2+-dependent cysteine proteases. Calpain-1 plays a crucial role in the chemotaxis of neutrophils and in activation of NFkB. It is, therefore, a potential drug target for the treatment of inflammatory disease. Calpains-1 & -2 „„

Have similar 3D structures yet inhibition of calpain-2 is undesirable. Hence, a thorough understanding of their structure and function is essential for future drug design. Domain structure „„

Both calpain isoforms form heterodimers. The large subunit contains an N-terminal a-helical domain that leads into the active site containing CysPc domain. The C2L domain containing an acidic loop follows leading to a regulatory Ca2+-binding PEF(L) domain. The small subunit consists of a glycine rich domain linked to another regulatory Ca2+-binding domain, PEF(S). The PEF(S) and PEF(L) domains form the main interface between the two subunits. Calpastatin „„

The calpain system is regulated in part by an endogenous inhibitor protein called calpastatin. Activation of calpain-1 & -2 „„

Both isoforms require binding of Ca2+ before they become active. Ca2+ concentrations spike at membranes where calpains are active but other factors contribute to activation of calpain. Ca2+ is thought to bind to the PEF domains followed by binding to the C2L and CysPc domains prior to autolysis of the a-helical domain. Thus, a conformational change occurs that brings together the catalytic dyad residues and activates the protease Physiological function & known substrates „„

The physiological substrates of calpains are unconfirmed but in vitro substrates include many cytoskeletal proteins. Calpains are thought to mediate many cellular process including cell-cycle progressions, apoptosis, activation of platelets and cell morphology changes. Inhibition of calpain „„

„„

Most inhibitors of calpain are general cysteine protease inhibitors that are not suitable as drug candidates. Some reversible biphenylbased inhibitors have been developed but have not been tested on both isoforms. A series of potent mercaptoacrylate inhibitors target the PEF domains and show some specificity for calpain-1. They inhibit the cell spreading activity of live neutrophils and so represent good leads for future drug development.

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