REVIEWS
Calpain research for drug discovery:
challenges and potential
Yasuko Ono1, Takaomi C. Saido2 and Hiroyuki Sorimachi1

Abstract | Calpains are a family of proteases that were scientifically recognized earlier thanproteasomes and caspases, but remain enigmatic. However, they are known to participate in a
multitude of physiological and pathological processes, performing ‘limited proteolysis’
whereby they do not destroy but rather modulate the functions of their substrates. Calpains
are therefore referred to as ‘modulator proteases’. Multidisciplinary research on calpains has
begun to elucidate their involvement in pathophysiological mechanisms. Therapeuticstrategies targeting malfunctions of calpains have been developed, driven primarily by
improvements in the specificity and bioavailability of calpain inhibitors. Here, we review thecalpain superfamily and calpain-related disorders, and discuss emerging calpain-targeted
therapeutic strategies.

Clan
Peptidases and theirproteinaceous inhibitors are
systematically classified by the
online MEROPS database.
A clan is composed of multiple
families, each of whichcorresponds to a group oforthologous peptidases.
Members of a clan sharesimilar primary and tertiary
structures. This classification
system complements andextends the previously
established enzyme
classification system, which
includes all of the enzymes.

Calpain Project, Departmentof Advanced Science for
Biomolecules, Tokyo
Metropolitan Institute ofMedical Science (IGAKUKEN),
2‑1‑6 Kamikitazawa,
Setagaya‑ku,
Tokyo 156–8506, Japan.
2
Laboratory for Proteolytic
Neuroscience, RIKEN Brain
Science Institute, 2–1
Hirosawa, Wako, Saitama
351–0198, Japan.
1

Correspondence to H.S. and
T.S. sorimachi‑hr@igakuken.
or.jp; saido@brain.riken.jp
doi:10.1038/nrd.2016.212
Published online DD Mmm 2016

We dedicate this Review to our late mentor Koichi
Suzuki, who made tremendous scientific contributions
as the world leader in calpain research for four
decades, led us into the world of calpains and died toosoon on 20 April 2010 aged 71 years.
Calpains, discovered in 1964 (REF. 1), are clan CA family C02 cysteine proteases (EC 3.4.22.17) defined by awell-conserved cysteine protease domain called theCysPc motif2–4 (FIG. 1). The word calpain combines ‘cal’ and‘ain’, which are respectively derived from calcium andcysteine proteases such as papain and legmain5. Calpains
were previously called Ca2+-activated neutral proteases6,7.
Although there are vast numbers of proteases andproteolytic complexes in biological systems, the calpains
are some of the very few that are directly activated by
Ca2+, a primary second messenger in signal transduction. In addition, calpains are modulator proteases that
perform limited proteolysis to modulate rather thanabolish the function of their substrate. Two key biological elements, Ca2+ and proteolysis, are embodied incalpains, and basic and clinical researchers often findthemselves trying to fit calpains into their subject ofinterest. The identification of calpains as aggravating
factors in human diseases can be attributed to such
scientific curiosity. However, although the vital rolesof calpains in various biological systems are clear, theirmechanistic features remain poorly understood. Owing
to their modulatory nature and lack of clear cleavage
sequence specificity, calpains appear to have less precise
activity and to be less amenable to systematic examination than proteases such as caspases and those involved

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in the ubiquitin–proteasome system. Consequently,
the biological functions and therapeutic potential of
calpains remain relatively unexplored.
Calpain-related disorders have a substantial impact
on society. Although some diseases are caused by genetic
defects, many are lifestyle-related8 or caused by infection
with a pathogenic microorganism9–13. These diseases areof growing concern worldwide from both human healthand economic perspectives. Disorders for which calpains
may serve as therapeutic targets include neurodegenerative disorders14–22, cardiovascular diseases23, ischaemic disorders24, arterial sclerosis25,26 and cancers27–30.
Additional disorders include cataracts31, muscular dystrophies32, gastric ulcer 33, vitreoretinopathy 34, oesophagitis35,36, pulmonary fibrosis37, diabetes38,39, malaria40,41,
trypanosomiasis42, schistosomiasis10, candidiasis43 andperiodontitis12. In most cases of calpain-related disease,calpain activity is elevated and aggravates symptoms. For
example, transient cerebral ischaemia caused by various
insults results in calpain activation that leads to neuronalcell death44.
Some calpain inhibitors have already been developed
for therapeutic applications. For example, the Aspergillusderived calpain inhibitor E-64d (also called loxistatin,
aloxistatin, rexostatine, EST and Estate) was used in clinical trials for Duchenne muscular dystrophy (DMD)45,
and the α‑ketoamide inhibitor A-705253 (also known as
BSF 419961 and CAL‑9961) has been tested as a treatment for Alzheimer disease (AD)46. More recently, it wasrecognized that restoration or compensation of calpain
activity could ameliorate diseases such as limb-girdle
muscular dystrophy type 2A (LGMD2A)47 and spastic
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REVIEWSa HumanProtease (CysPc) domain
Cys

Conventional calpain subunits:• CAPN1 or CAPN2 (also known as μCL or mCL, respectively)• CAPNS1 (also known as 30K)

kDa

His Asn
80
+
28

GR

Unconventional classical calpain subunits:• CAPN3 (also known as p94)
• CAPN8 (also known as nCL-2)• CAPN9 (also known as nCL-4)• CAPN11–14

IS1
Cys

IS2

94

His Asn80

Cys/Lys His Asn

Unconventional non-classical calpain subunits:• CAPN5 (also known as hTRA3)• CAPN6 (also known as CANPX)C2Cys• CAPN7 (also known as PalBH)• CAPN10(75

His Asn)93, 75Cys

His Asn120• CAPN15 (also known as SOLH)
Cys
IQ• CAPN16 (also known as demi-calpain)

CC

190

b Schistosoma mansoni
Cys

His AsnCys

His Asn• Smp-157500

180

c Plasmodium falciparum

Cys

His Asn• Pf-calpain

230

d Trypanosoma brucei• ClpGM6
Cys

Gln AsnCys

Gln Asn>70 GM6 repeats

Gly

Gln Asn
820

Ser• CAP5.5 (also known as TbCALP1)

Tyr Asn95

ABS

e Aspergillus nidulans, Saccharomyces cerevisiae

Cys(• PalB/Rim13

f Porphyromonas gingivalis

His Asn

)

94/83
Cys

His Asn• Tpr

55
300 aarN region

PC1

PC2

Figure 1 | Schematic structure of calpains. a | In vertebrates, calpain-1 andcalpain-2 are heterodimers of a large catalytic subunit (CAPN1 and CAPN2,
respectively) and a small regulatory subunit (CAPNS1). Calpain-1 andcalpain-2 are called conventional calpains, with the remaining calpains
known as unconventional calpains (avian calpain-11 (CAPN11–CAPNS1)
may also be called a conventional calpain). Calpains are classified into two
types on the basis of their structure: classical, with domain structures thatare identical to those of CAPN1 and CAPN2, and non-classical. b–f | Amongthe non-human calpains shown, only Smp‑157500 (the carboxyl-terminal
half is known as Smp-80) of Schistosoma mansoni is a classical calpain.

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CBSWPEF(L)

PEF(S)

MIT

Zn

SOH

GM6

Plasmodium falciparum and Porphyromonas gingivalis each have only one
calpain, whereas S. mansoni, Trypanosoma brucei and Aspergillus nidulans
Nature| Drug
Discovery
have 7, 18 and 2 calpains in total, respectively.
TheReviews
number of
repetitionsof
GM6 antigen repeat unit could not be determined owing to an incomplete
genomic sequence. ABS, all β‑strand structure (found among several
glycosyl hydrolases); CBSW, calpain-type β‑sandwich domain; CysPc,
cysteine protease domain, calpain-type; GR, glycine-rich domain; IQ, calmodulin-interacting domain; IS, insertion sequence; MIT, microtubule-interacting and transport domain; PC, protease core; PEF, penta‑EF‑handdomain; SOH, SOL-homology domain; Zn, zinc-finger motif.

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CysPc motif
A protease catalytic domain of
calpains. CysPc-containing
proteases belong to the sameclan as papain, but unlike thecatalytic domain of papain the
CysPc divides into two separate
structures, protease core 1(PC1) and PC2, in the absenceof Ca2+. Amino acid sequencecomparisons suggest that all of
the calpain species for which
3D structures are not yet
solved share similarity in theirCysPc domain.

α‑Ketoamide inhibitor
A class of reversible inhibitors
for cysteine, serine or
threonine proteases that add
an electrophile to these active
site amino acid residues. Many
inhibitors of this class havebeen developed bysystematically replacing
aldehyde moieties of known
calpain inhibitors with
α‑ketoamide, andsubsequently modifying otherpositions to improve
effectiveness.

Calpainopathies
Diseases caused by a genetic
defect in a calpain gene. The
pathogenic mechanism can beeither loss of function (forexample, limb-girdle muscular
dystrophy type 2A is caused
by inactivating mutations in the
CAPN3 gene) or gain offunction (for example,
autosomal dominantneovascular inflammatory
vitreoretinopathy is caused by
an excessive activation of
CAPN5 due to mutations).

Calpain-type β-sandwich(CBSW). A domain whose 3D
structure, but not primary
sequence, shows overallsimilarity to the C2 domain, acalcium-binding motif found inprotein kinase C,
synaptotagmins and othercalcium-related proteins. The
CBSW domain has a role insubstrate recognition.

Penta‑EF‑hand(PEF). Among the proteins with
Ca2+-binding EF‑hand motifs,
those with five EF‑hand motifs
in tandem comprise the PEF
family. The fifth EF region isoften involved in homo- or
heterodimerization. Classicalcalpains have a PEF domain,and hence also belong to the
PEF family.

paraplegia48. Therefore, calpain-targeting strategies now
encompass two key approaches: calpain inhibition andcalpain activation (either directly or by replacement).
This Review seeks to address how the wealth of
discoveries gained from calpain research can be translated into strategies for combating various disorders.
In this regard, we first describe the current state of
calpain research and discuss the pathological roles of
calpain activity. We then assess various therapeuticstrategies that target calpains as defined by three major
approaches: first, the inhibition of human calpains that
aggravate symptoms; second, inhibition of the calpainsof parasites or pathogenic microorganisms to disrupt theinvasion, growth and/or egress of the organism; and, last,complementation of calpain functions by gene therapy
or gene editing to correct or compensate for a defective
calpain. Importantly, the challenges faced in therapeutic
modulation of calpain activity are discussed.

The calpain superfamily
Human calpains. Calpains exist in almost all eukaryotes
and some bacteria, whereas calpains are not present invery few fungi and no calpain gene has been found so
far in the genome of any archaea49–52. Various motifs or
domains (>40) occur together with the CysPc motif 51,52.
Humans express 15 calpain genes, CAPN1 to CAPN16
except for CAPN4, each of which generates one or moretranscripts and constitute a superfamily of more than 50
molecular species (for example, CAPN3 has more than
10 variants)4,53. Mutations in individual calpain genes
are associated with lethality or disorders known as cal‑painopathies, indicating the importance of calpains inmammalian life and health4,53 (see below and TABLE 1).
In non-mammals, calpains have also been geneticallyshown to be involved in various biological phenomena, such as optic neuron development in fruit flies, sex
determination in nematodes, alkaline environment adaptation in fungi and yeasts, and germ cell generation inplants2,4,49,54,55.
Calpain-1 and calpain-2, known as the conventional calpains, are the most ubiquitous and well-studied calpain family members, and are activated in vitro
by micromolar and millimolar concentrations of Ca2+,
respectively 2. They modulate the structures and functions of their substrates by limited proteolysis2,50. Thereis a long-standing paradox regarding the mechanisms of
calpain-1 (also known as μ-calpain) and calpain-2 (alsoknown as m‑calpain)activation: given that physiological intracellular Ca2+ concentrations reach micromolar
levels at most, calpain-1 was thought to be used mostwidely in vivo. However, reverse genetics has shown that
CAPN1‑deficient mice are apparently normal56, whereas
CAPN2 deficiency induces embryonic lethality 57,58, suggesting that calpain-2 has more important functions
than calpain-1 in vivo. The reason for the discrepancy
between the biochemistry and the biology of calpain-1
and calpain-2 remains largely elusive.
Among the unconventional calpain subunits, CAPN5,7, 10, 13 and 15 also exist in most cells (called ubiquitous
calpains for descriptive purposes), whereas CAPN3, 6,
8, 9, 11, 12, 14 and 16 are expressed predominantly in

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particular tissues and organs (tissue-specific calpains)4,50.
The tissue-specific calpains, when defective, cause disorders specific to the tissues in which they are expressed.
For example, pathogenic mutations in CAPN3, which
is predominately expressed in skeletal muscle, result in
LGMD2A32 (TABLE 1). By contrast, the ectopic expressionof CAPN3 in the heart, another tissue composed of striated muscle, is lethal, highlighting the need for tightly
regulating calpain expression in specific tissue and cellular environments47,59. Intriguingly, despite their presence
in most tissues, pathogenic alterations in CAPN1 andCAPN5 lead to deficiencies in specific tissues (neuronalcells and eyes, respectively)34,48.
Between 1964 and 1988, calpain research focusedsolely on conventional calpains. The first unconventional calpain was identified in 1989 (REF. 60), followed by
the discoveries of others. Since the twenty-first century,
unconventional calpains have become a critical topic
in the calpain field4,50. An endogenous proteinaceous
calpain inhibitor, calpastatin (CAST), has been foundin mammals and birds, and adds another layer of complexity to the calpain system61. The inhibitory activity of
CAST is specific for calpains, and it does not inhibit anyother proteases61 (see below).
Structures of calpains. The conventional calpains consist of a large catalytic subunit and a small regulatory
subunit (FIG. 1). The large subunit (CAPN1 and CAPN2
for calpain-1 and calpain-2, respectively) has an amino‑terminal anchor helix (N region), a CysPc domaincomposed of protease core 1 (PC1) and PC2 domains,
a calpain-type β‑sandwich (CBSW; previously known as
C2‑domain-like (C2L)) domain, and a penta‑EF‑hand(PEF)62 domain (PEF(L)). The common small subunit
CAPNS1 is composed of glycine-rich (GR) and PEF(S)domains (FIG. 1) and is essential for the stability of CAPN1
and CAPN2. Genetic disruption of Capns1 in mice inactivates both calpain-1 and calpain-2 (REFS 58,63). CAPN3,8, 9 and 11–14 have domain structures identical to thoseof CAPN1 and 2, and are called classical calpain subunits, whereas the remaining non-classical calpain subunits have another motif (or motifs) in place of the PEF
domain. CAST, the calpain-specific inhibitor protein, has
several splicing variants, the longest of which consists of
N‑terminal non-inhibitory regions and four repeatedcalpain-inhibitory domains, which are each composedof three subdomains: A, B and C61. Subdomain B interacts with the CysPc domain, whereas subdomains A andC, which form α‑helices, bind to PEF(L) and PEF(S) of
the conventional calpains, respectively, and stabilize the
CAST–calpain complex 64,65.
The 3D structure of calpain-2 was the first to bereported64–67. So far, the only calpain for which the complete 3D structure has been solved is calpain-2; however,
3D modelling of other calpains has been accepted as arational approach68–71. Four domains of CAPN2 alignon the same surface, which spreads over an imaginary
oval plane. In the absence of Ca2+, PC1 and PC2 haveopen structures that do not form a catalytic triad66,67.
Activation by Ca2+, surprisingly, does not dramaticallychange the overall calpain structure; instead, one Ca2+ADVANCE ONLINE PUBLICATION | 3

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Table 1 | Calpainopathies
Gene (alternative names)*

Expression preference

Phenotype related to gene mutation

CAPN1, Capn1

All cells

Spastic paraplegia48, platelet dysfunction56 andspinocerebellar ataxia (hereditary also in dogs)170,171

Capn2

Most cells

Embryonic lethal57,58

Capns1All cells

Embryonic lethal63,263

CAPN3, Capn3

Skeletal muscle

Various muscular dystrophies32,161,264,265

CAPN5, Capn5

Most cells

Vitreoretinopathy34,165

Capn6

Embryonic muscle

Hypergenesis240

CAPN7

Most cells

NP

Capn8

Gastrointestinal tract

Gastric ulcer33

Capn9

Gastrointestinal tract

Gastric ulcer33

CAPN10

Most cells

Type 2 diabetes251

CAPN11

TestesNP

CAPN12

Hair follicles, skin

Congenital ichthyosis266

CAPN13

Most cells

NP

CAPN14

Oesophagus

Eosinophilic oesophagitis35

CAPN15 (SOLH)

Most cells

NP

CAPN16 (ADGB, C6ORF103)

TestesNP

NP, not yet published for human or mouse gene. *CAPN and Capn indicate human and mouse genes, respectively.

CAPNS1(Calpain small subunit 1).
A PEF family member.
A paralogue called CAPNS2
has an unknown function.
In vivo, CAPNS1 is an essential
component of the twoconventional calpains,
calpain-1 and calpain-2.
However, in vitro, the cysteine
protease domains isolated
from CAPN1 and CAPN2(mini-calpains) are functional asactive proteases.

binds to each of the PC domains, which causes PC1 andPC2 to connect, forming the ‘closed’ active structure64,65(FIG. 2). Crystal structures of the active calpain-2–CAST
complex show that the CAST amino acid residues thatinteract near the active site of calpain do so mostly
through their peptide bond backbone atoms, whereas
the side chains of CAST are mostly oriented outwards
from the structure and do not engage in atomic interactions with calpain64,65. This preference for binding to the
peptide bond backbone atoms may explain the apparently low amino acid sequence selectivity for calpain
substrates.
The cleavage site of a protease substrate can be
described as the bond between amino acid site P1 on the
N‑terminal side and P1′ on the carboxy‑terminal side.
The residues oriented away from the cleavage site arelabelled P2, P3, and so on, and P2′, P3′ and so on. The
site in the binding pocket of the protease that binds with
P1 is called S1, and so on. In calpain substrates, P2, P1
and the primed sites interact with CysPc, whereas mostof the unprimed sites in the N‑terminal to P2 are recognized by CBSW64,65 (FIG. 2). Together with the 3D structures of CysPc–inhibitor complexes72–75, these findings
indicate that the P2–S2 interaction between a conventional calpain and its substrate is prominent, similar toother clan CA proteases such as cysteine cathepsins, andthat the primed sites are also important for the efficientand specific blockade of calpains by inhibitors.
A recent structural insight worth noting is that of
the complex of calpain with its allosteric inhibitors. To
date, an α‑mercaptoacrylic acid derivative (PD150606)
and an oligopeptide (LSEAL) have been proposed toexert an allosteric inhibitory effect on calpains76–78. Bothmolecules bind to hydrophobic clefts in the calpain PEF
domains in a manner similar to the CAST subdomains

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A and C. However, a recent study showed that PD150606
inhibits calpains by directly interacting with the CysPc
domain, and that LSEAL does not inhibit calpains
in vitro79. As discussed below, the mode of inhibition by
PD150606 remains elusive.
The 3D structure of calpain has revealed new strategies for inhibitor design. For example, the specific inhibition of calpain by CAST is enabled by the core inhibitory
sequence within subdomain B of CAST, which is positioned beyond the active site64,65. This finding has led toa new concept for inhibitor design, in which molecules
such as macrocyclic peptides are of interest. In addition, although the available structural data are currently
limited, examining differences among the CysPc structures from different calpains is possible: for example,
the domain rotations of PC1 and PC2 relative to eachother 72,80,81 (FIG. 2). Such information can direct the designof specific inhibitors and/or substrates for each calpain
species (BOX 1).
Substrate specificities of calpains. The identificationof calpain substrates has revealed several signal transduction-related molecules for which proteolysis by calpain results in functional alterations. One interesting
substrate is p35, which can be converted by calpain to
p25, an activator of cyclin-dependent kinase 5 (CDK5)
that may be involved in memory formation82. Anotherinteresting substrate is interleukin‑1α (IL‑1α)83, an
inflammation-related cytokine in humans. IL‑1β issecreted after its cleavage by caspase 1, whereas IL‑1α
is cleaved by calpain84 and then transported out of thecytoplasm by an as yet unknown mechanism. In addition, cleavage of the MYC oncoprotein by calpain is a
critical step in transforming the functions and localization of this key molecule in cancer cell survival85–87. The
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PC2

Leupeptin
Activesite

CAST(P1 P10)CAST(P10ʹ P1ʹ)

PC1
CBSW

CAPN1, Ca2+
CAPN8, Ca2+

CAPN2, Ca2+
CAPN9, Ca2+

CAPN2, Ca2+
CAST, Ca2+

Figure 2 | Cross-eye stereo view of CAPN1, 2, 8 and 9. The cysteine protease (CysPc) domain of CAPN1, the large
catalytic subunit of calpain-1 (RCSB Protein Data Bank (PDB) ID: 1TL9), CAPN8 (PDB ID: 2NQA) and CAPN9 (PDB ID: 2P0R)
are shown in complex with leupeptin (shown by stick models) and Ca2+ (shown by balls). The
CycPc
and calpain-type
Nature
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β‑sandwich (CBSW) domains of CAPN2 (PDB ID: 3BOW (red) and 3DF0 (pink)) in a complex with calpastatin (CAST;
indicated by dark green tubes; right to left corresponds to amino terminus to carboxyl terminus) and Ca2+ weresuperimposed at the protease core 1 (PC1) and PC2 regions. The amino acid alignment (data not shown) according tothese superimpositions indicates that the amino acid residues proximal to CAST are well conserved among CAPN1, 2, 3, 8
and 9, except that several amino acid residues in CBSW (corresponding to S3–S10) are divergent in CAPN3. Structuresgenerated by MOE Ver.2015.10 (Chemical Computing Group Inc., Montreal, Quebec, Canada).

calpain-mediated cleavage of dysferlin, a gene productresponsible for LGMD2B, also exemplifies the transformative nature of calpain. The C‑terminal C2 domainsof dysferlin are released by calpain in response to membrane injury, and these domains function as effectors
in a membrane repair cascade88,89. Finally, one of thebest-known calpain substrates is spectrin, an important
membrane cytoskeletal maintenance protein (spectrin‑α
chain non-erythrocytic 1 is also known as α‑fodrin). The
calpain-dependent proteolysis of spectrin is closely associated with neuronal cell death90. Spectrin is proteolysedat a specific site by calpain-1 and calpain-2 (REFS 91,92),
as well as by other calpains93.
Elucidating substrate specificity at the molecularlevel has been a challenge in calpain research, although
it is emerging as an attractive subject for bioinformatics studies. Initial studies at the protein and amino acidsequence levels revealed the limited nature of the proteolytic activity of conventional calpains94. A few years later,a position-specific scoring matrix analysis revealed theamino acid residue preference at each position aroundthe cleavage site, which provided the first theoreticalinformation regarding the substrate specificities of
calpains95. Further studies were carried out using peptide libraries74,96–98 and/or machine learning 98–102, whichyielded calpain cleavage site predictors that were sufficiently accurate for practical use98–102. A comprehensive
kinetics analysis of peptide-array substrates showed thatin addition to P2, the P3′ and P4′ sites are important forthe cleavage efficiency of calpains98. These features may
provide directions for peptide-based inhibitor design.
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Human disorders involving calpains
Calpain-associated diseases can be categorized into three
types according to the way calpain is involved in theirpathogenesis: those caused or exacerbated by humancalpain activities (type 1; for example, neurodegenerative and cardiovascular disorders, ischaemia, cancers andcataracts); those caused by parasites or pathogenic microorganisms that use the host’s and/or their own calpains
for infection and survival (type 2; for example, malaria,
trypanosomiasis, schistosomiasis, candidiasis and periodontitis); and calpainopathies caused by deficiencies
in calpain genes (type 3; for example, muscular dystrophies, vitreoretinopathy, gastric ulcer and oesophagitis)(TABLES 1,2). Representative examples are described below.
Many studies of calpain-related disorders have usedtransgenic mice. For example, mice deficient in calpain-1
and/or calpain-2 and mutant mice that lack or overexpress CAST (Cast−/− and transgenic CAST mice, respectively)23,59,103–105. Together with antibodies that recognize
activated CAPN1 (REF. 106) or calpain-cleaved spectrin107,
these mouse models have been valuable tools for revealing clear cause-and-effect relationships in neurodegenerative and other disorders. However, there is growing
awareness that transgenic mice are an idealized and simplified experimental system and that the results obtainedusing these systems must be carefully interpreted.
Neurodegenerative disorders. Several reports have indicated that calpain may have a major aggravating role in
the pathogenesis of AD20,108–110. For example, calpain-1 is
hyperactivated in the AD brain108, and calpain inhibitors
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Box 1 | Improving the specificity of human calpain inhibitors
Most calpain inhibitors (Supplementary information S1,S2 (figure, table)) are not specific and block other proteases suchas cysteine cathepsins. Calpastatin (CAST) is the only absolutely specific inhibitor for classical calpains: calpain-1(CAPN1–CAPNS1) and calpain-2 (CAPN2–CAPNS1), calpain-9 (CAPN9–CAPNS1), calpain-8 (CAPN8)n) and calpain-8/9(CAPN8–CAPN9; also known as G-calpain)2,33,61,253; however, it does not inhibit calpain-3 (CAPN3)68,254. The mini-calpain
mutants cysteine protease (CysPc)-only CAPN1 and CAPN2 are also not effectively inhibited by CAST255,256. The calpain
amino acid residues proximal to the bound CAST are well conserved among CAPN1, 2, 3, 8 and 9, except that severalamino acid residues of the calpain-type β-sandwich (CBSW) domain that contact CAST at the P3–P10 sites are divergent
in CAPN3. The alignment of the CBSW domain relative to the CysPc domain may also be different between CAPN3 andother calpain subunits. Thus, the CAST–CBSW interaction appears to be key for CAST specificity.
The existence of CAST indicates that there is a solution to developing absolutely specific calpain inhibitors. A relatively
long peptide bridging CysPc and CBSW may be required. Once an inhibitor is discovered, issues such as its stability andbioavailability in vivo, membrane penetrability and production cost need to be examined. With regard to these issues,secondary-structure-fixed inhibitors (CAST peptidomimetics and macrocyclic peptides) discussed in the main text188,190,192
and calpain-derived inhibitory peptide193 appear to be promising. Currently, although the specificity of these peptide
inhibitors is satisfactory, their stability in vivo, bioavailability, cost to synthesize and/or cell penetrability need to be
improved for their application as therapeutic agents. Among these inhibitors, β‑strand-fixed macrocyclic inhibitors canbe relatively easily applied for proteases other than calpains (such as cathepsins L and S, and proteasomes) by
substituting amino acid residues191. This approach may be applied for isoform-specific calpain inhibitors (see below).
Almost all calpain inhibitors are active site-directed, which is one of the reasons for their poor specificity for calpains.
Because protease core 1 (PC1) and PC2 need to connect to be active, inhibiting this connection, for example, by anintercalation between PC1 and PC2, may result in supremely specific inhibitors for calpains. Similarly, other allostericinhibitors discussed in the main text are also worth pursuing79,195,196. Alternatively, when the involvement of cysteine
proteases other than calpains cannot be ruled out owing to a lack of inhibitor specificity, inhibitors specific for cysteine
cathepsins can be tested for their ability to suppress disorders that are ameliorated by nonspecific calpain inhibitors.
Specific inhibitors for cathepsins B, K, S and L are available (CA‑074, odanacatib, CLIK‑060 and CLIK-148,
respectively)247,248. Comparing the effects of these cathepsin inhibitors with those of calpain inhibitors such as ALLNal and
MDL28170 may help to identify the responsible protease (or proteases) for various phenomena. Specifically targeting
inhibitors by conjugating them with molecules with an affinity for particular sites or tissues — for example, carnitine(muscle)127, taurine (neuron)214 or pregabalin (brain)215 — is another promising method.
At the molecular level, some differences in the substrate specificities of calpain-1 and calpain-2 have been recently
revealed98, and distinct biological functions of these calpains have also been reported150,257–260. Thus, differential
inhibitions of calpain-1 and calpain-2 may improve the efficacy of some calpain-targeted drugs. One of the peptidyl
α‑ketoamide derivatives shows more than 100‑fold selectivity for calpain-2 over calpain-1 (mCalp‑I; Supplementary
information S2 (table))261,262. Although a calpain-1‑specific inhibitor has not yet been found (PD151746 shows only modest
selectivity for calpain-1 over calpain-2, approximately 20‑fold; see Supplementary information S2 (table)), the existenceof mCalp‑I indicates that such inhibitors are worth seeking.

can improve memory and synaptic function in miceoverexpressing the amyloid precursor protein (APP), a
model of AD109. This view was supported by findings that
CAST deficiency induces the hyperphosphorylation oftau, somatodendritic atrophy, early lethality, augmented
amyloid-β (Aβ) amyloidosis and elevated neuroinflammation in APP-overexpressing mice109. However, mostof these phenotypes could not be reproduced using asecond-generation mouse model of AD, which accumulates Aβ42 without overexpressing APP; thus, the rolefor calpain in AD pathogenesis was challenged20,110. A
recent study has shown that overexpression of APP andpresenilin in mice causes calpain activation, indicatingthat preclinical studies should not unduly rely on overexpression paradigms111. Nevertheless, CAST deficiency
accelerates Aβ amyloidosis and neuroinflammation in
the second-generation mouse model, indicating that thecalpain–CAST system may regulate the fundamental
pathological processes of AD20,110.
Calpain activity also appears to be an important
player in other neurodegenerative diseases. For example,
CAST overexpression reduces protein aggregation andsynaptic impairment in mouse models of Parkinson disease (PD)19. In addition, the calpain-dependent cleavage
6 | ADVANCE ONLINE PUBLICATION

of TDP43, a 43 kDa heterogeneous nuclear ribonucleoprotein that binds to the TAR DNA sequence and regulates transcription and RNA splicing, is an essential
contributor to amyotrophic lateral sclerosis (ALS) aetiology 15. Furthermore, CAST deficiency aggravates thepathology and symptoms of animal models of spinocerebellar ataxia type 3 (REF. 16).
Calpain inhibition may also be therapeutically beneficial in lissencephaly, a developmental brain disordercaused by defective neuronal migration14. Turnover of
one of the responsible gene products, LIS1, is regulated
by calpain proteolysis, and a heterozygous loss of LIS1
results in insufficient amounts of LIS1 protein, causing
defective intracellular microtubule network (homozygous loss results in lethality)14. Administration of calpain
inhibitors to Lis1+/− mice (a model of lissencephaly) after
disease onset significantly rescued defects in brain functions related to motor behaviour and glucose metabolism14,17. Calpain inhibitors tested in Lis1+/− mice include
small interfering RNA (siRNA) targeted against Capns1,
ALLNal (also known as calpain inhibitor I; an inhibitorof calpain-1 and calpain-2 and cysteine cathepsins) andSNJ1945 (a cell-permeable second-generation inhibitorof calpain-1 and calpain-2)14,17.
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Table 2 | Diseases involving calpains and their therapeutic strategies
DiseaseEffect of activity

Calpain-related
therapeutic candidates

Calpain-1 and -2 (human, mouse)

Aggravating or causative

Calpain inhibitors

Related calpain protein or
gene (source)

Refs

Disease with a primary cause other than calpains
Atherosclerosis

25

Brain ischaemia

Calpain-1 and -2 (human, mouse)

Aggravating or causative

Calpain inhibitors

24,44

Cardiovascular disorders

Calpain-1 and -2 (human, mouse)

Aggravating or causative

Calpain inhibitors

23,39,114,116,
117,119

Cataracts

Calpain-1 and -2 (human, mouse)

Aggravating or causative

Calpain inhibitors

92,134,136–139

Neurodegenerative disorders (e.g.
Parkinson disease, amyotrophic
lateral sclerosis, spinocerebellar
ataxia type 3 and lissencephaly)

Calpain-1 and -2 (human, mouse)

Aggravating or causative

Calpain inhibitors

14–17,19

Retinitis pigmentosa

Calpain-1 and -2 (human, mouse)

Aggravating or causative

Calpain inhibitors

Cancers

Calpain-1, -2 CAPN3 and CAPN9(human, mouse)

Preventive, aggravatingor causative

Calpain inhibitors and/or
gene therapy

Alzheimer disease

Calpain-1 and -2 (human, mouse)

May be aggravating or
causative

Potentially calpain
inhibitors

20,110Muscular dystrophies

Calpain-1 and -2 (human, mouse)

May be aggravating or
causative

Potentially calpain
inhibitors

130

Cardiac scar

Calpain-1 and -2 (human, mouse)

Preventive

Potentially gene therapy

120

Dilated cardiomyopathy

Calpain-1 and -2 (human, mouse)

Preventive

Potentially gene therapy

121

Fungal infection (opportunistic
infection)

PalB/Rim13 (fungus)

Aggravating or causative

Calpain inhibitors

181,182

Malaria

Calpain-1 and -2, Pf‑calpain(human*, mouse*, parasite)

Aggravating or causative

Calpain inhibitors

41,178Periodontitis

Tpr (bacteria)

Aggravating or causative

Calpain inhibitors

12

Trypanosomiasis and leishmaniasis(e.g. African sleeping sickness)

Calpain-1 and -2, ClpGM6(human*, mouse*, parasite)

Aggravating or causative

Calpain inhibitors

180

Schistosomiasis

Smp‑157500 (parasite)

Aggravating or causative

Vaccine (rSm‑p80)

10,225

CAPN5 (human, mouse)

Aggravating or causative

Calpain inhibitors

34,167141,142
29,85,86,145,
147–159

Disease model

Infectious disease

Calpainopathies
Autosomal dominant neovascularinflammatory vitreoretinopathy
Eosinophilic oesophagitis

CAPN14 (human, mouse)

Preventive

Potentially gene therapy

35,36

Gastric ulcer

CAPN8, CAPN9 (human, mouse)

Preventive

Potentially gene therapy

33

Limb-girdle muscular dystrophy
type 2A

CAPN3 (human, mouse)

Preventive

Potentially gene therapy

32

Spastic paraplegia 76

CAPN1 (human, mouse)

Preventive

Potentially gene therapy

48

Type 2 diabetes

CAPN10 (human, mouse)

Not definable

Unclear251,252

Pf, Plasmodium falciparum; rSm‑p80, recombinant Smp‑157500 (carboxy‑terminal 80 kDa fragment). *Calpains in host cells — that is, human or mouse — are used by
the parasite.

Brain ischaemia. Brain ischaemia is caused by various
insults, including cerebral infarction, haemorrhage,vasospasm and, sometimes, cardiac infarction. Brainischaemia results in neuronal glucose starvation, ATPdepletion and a rise in Ca2+ levels44. Transient cerebral ischaemia in gerbils is accompanied by calpain
activation, which occurs in two phases: an immediate
activation of calpain in hippocampal CA3 neurons followed by substantial calpain activation in CA1 neurons
approximately 7 days later 112. This process results in an
overall delay in cell death of CA1 neurons, which can
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be blocked by administering the experimental calpain
inhibitor ALLNal24.
However, calpain is not easy to activate under physiological conditions. For example, almost lethal doses of
kainic acid are required to achieve the forced activationof brain calpain, even in Cast−/− mice105. The observationthat calpain is not widely activated in the brain supports
the idea that if spatiotemporally restricted in the brain
for a short period, a calpain-specific inhibitor would
generate only minor side effects on physiological processes, at least in the experimental context.
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REVIEWS
Cardiovascular disorders. One of the pathologicalmechanisms underlying many acute cardiovasculardisorders8,113, including ischaemia–reperfusion andpressure-overload models, is thought to involve the calpain-mediated proteolysis of myocardial proteins114,115.
Accordingly, calpain inhibitors are able to ameliorate
symptoms of these disorders in animal models (TABLE 3).
A protective effect of ALLNal, but not of a broad caspase
inhibitor, on the myocardial injury and mitochondrial
dysfunction caused by ischaemia–reperfusion revealed
that calpain activation can result in cell death independent of caspases116. More specifically, mitochondrial calpain-1 was suggested to be involved in cardiac injury 117;
this injury could be prevented by the calpain inhibitor
MDL28170, a cell-penetrating dipeptidyl aldehyde inhibitor that also acts on cysteine cathepsins118. MDL28170
is also called calpain inhibitor III (see Supplementary
information S1,S2 (figure, table)).
Another calpain inhibitor, A-705253, a benzoylalanine-derived α‑ketoamide inhibitor with improved
water solubility, metabolic stability and cell permeability,
exhibited protective effects on heart functions and global
haemodynamics in a porcine myocardial ischaemia–reperfusion model119. Results from mice in which calpain
activity was genetically inhibited by transgenic CAST
expression or Capns1 disruption also suggested that
calpain is an aggravating factor in the chronic cardiac
complications associated with type 1 diabetes or angiotensin II infusion23,39. Furthermore, the calpain inhibitors
ALLMal, calpeptin and BDA‑410 suppress atherosclerosis in Ldlr−/−, Apoe−/− and angiotensin II‑treated Ldlr−/−
mice by blocking the proteolysis of vascular endothelial
cadherin and/or spectrin25,26 (TABLE 3).
Although calpain inhibition appears to be a promising approach for the treatment of several cardiovascular disorders, some adverse effects of calpain inhibition
have been reported. For example, dilated cardiomyopathy and impaired scar healing are observed in transgenic
CAST mice, in which endogenous calpain activity is
reduced59,120. In addition, cardiac-specific Capns1−/− mice,although healthy under normal conditions, show cardiac
dysfunction under pressure overload121. These studies
suggest that calpain activation is required under somepathological conditions to overcome cardiotoxicity.
One possible mechanism by which calpain protects heart function is related to its role in membrane
repair 89,122. Meanwhile, studies using human blood
cells and mouse models have indicated that calpain
has opposing effects on inflammation and immune
processes depending on the context, such as its localization123. In addition, an upregulation of CAPN2 wasassociated with the effect of a potential cardioprotective
reagent, which is being considered for use in combination chemotherapeutic drug therapy 124. Further clarification of the precise targets and timing of calpain activity
will help us to evaluate the balance between the possible
benefits and limitations of calpain inhibition.
Myopathies. In DMD, the activities of conventional
calpains are upregulated due to a loss of Ca2+ homeostasis2,113. Several muscle proteins are substrates of
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calpains; thus, increased calpain activity is thought toexacerbate symptoms. In fact, studies using the dystrophin-deficient DMD mouse model, mdx mice, demonstrated that the increased calpain activation in necrotic
muscle fibres was corrected in CAST overexpression
in mdx mice, accompanied by the amelioration of thedystrophic phenotype103,125. Thus, proposed pharmacological approaches for ameliorating DMD have longincluded calpain inhibition. For example, the ability of
inhibitors such as E-64d and leupeptin to delay muscle degeneration has been extensively studied45,126 (seebelow). Promising results in model animals have led tofurther studies seeking to improve the bioavailability ofthese inhibitors in skeletal muscle tissues127.
More recent studies using mdx mice128 and a golden
retriever DMD model129, however, did not support thelong-standing view that the pharmacological inhibitionof calpains ameliorates DMD pathology. These studies
showed that a decrease in calpain activity does not necessarily improve muscle functions, and also warned thatchronic calpain inhibition by either inhibitor administration or CAST overexpression induces a compensatory
upregulation of calpains. Thus, the outcomes of suchtreatments or conditions need to be carefully and consistently analysed130.
Meanwhile, α‑klotho, defects in which causes senescence, is specifically suppressed in the muscles of mdx
mice after the clinical onset of muscular dystrophy, andtransgenic α‑klotho expression rescues the muscle atrophy phenotype in these mice131. The α‑klotho protein
modulates Ca2+ homeostasis, and Kl−/− mice exhibit
overactivated calpain-1 and phenotypes reminiscent ofageing-related disorders. Such phenotypes include infertility, body weight loss, multiple organ atrophies, ectopic
calcification and bone mineral density reduction132.
These symptoms are ameliorated by the administrationof the calpain inhibitor BDA‑410 (REF. 133), implicating
a role for α‑klotho in regulating calpain activity (TABLE 3).
Downregulation of α‑klotho is also found in muscle
biopsies from patients with DMD131, suggesting that calpain inhibition might be more therapeutically effective if
combined with another treatment that compensates fora deficiency in α‑klotho function.
Ophthalmic diseases. Cataracts, a prominent age-related disease, result from the aggregation of crystallines,
which is caused by oxidative damage and/or by over-proteolysis by calpains and other proteases31. Calpain-2 has
a major role in human cataractogenesis by proteolysing
the major lens proteins α- and β-crystallins134, whereas
variants of CAPN3, such as Lp82, and CAPN10 are also
involved, especially in rodent models135. In transgenic
mice expressing K6W mutant ubiquitin in the lens, thealtered ubiquitin–proteasome system causes calpain
hyperactivation, resulting in disease progression136.
Thus, the development and application of calpain inhibitors for the treatment of cataracts has been an active
topic in calpain research. In vitro, a preventive effect of
calpain inhibitors against cataractogenesis in cultured
rat lenses was first demonstrated for E-64 (REF. 137),
and later for SJA6017 (REF. 92), a more potent calpain
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Table 3 | Selected in vitro and in vivo studies (since 2010) of calpain inhibition in disease
Inhibitor or vaccine

Disease/phenomenon

Model

Target*Effects

ALLNalCoxsackievirus
B3 infection andreplication

H9c2 cells

Calpains• ↓ Virus titre• ↓ Autophagy

11

Uterine implantation

Mice

Calpains(CAPN2)• ↓ Implantation

246

Lissencephaly

Lis1+/− mice

Calpains• No change in LIS1 cleavage• ↓ Spectrin-α cleavage• ↓ Hyperexcitability (spontaneous andminiature excitatory postsynaptic current)

267

ALLNal, PD150606

Retinitis pigmentosa

Royal College ofSurgeon’s rats

Calpains• ↓ Retinal cell apoptosis

268

ALLNal, calpeptin

Oestrogenmediated cancer
metastasis

Oestrogen andpure anti-oestrogen
fulvestrant (ICI 182780)-treated MCF‑7
cells

CAPN1

• ↓ Cell–Matrigel adhesion

269

Calpeptin

Parkinson disease

MPTP-induced acute
Parkinsonian mice

Calpains• ↓ Glial activation• ↓ T cell infiltration• ↓ Neuronal death• ↓ Gait deficit

270

Multiple sclerosis

EAE in Lewis rats

Calpains• ↓ Gliosis• ↓ Loss of myelin• ↓ Axonal damage• ↓ Inflammation and microgliosis in optic nerve

271,
272

Idiopathic
inflammatory
myopathies

IFNγ- or TNF-treated
rat L6 myoblasts

Calpains• ↓ MHC class I- and inflammation-relatedtranscription factors• ↓ Apoptosis

273

Idiopathic pulmonary
fibrosis

Bleomycin-induced
pulmonary fibrosis inmice

Calpains• ↓ Lung fibrosis• ↓ Bleomycin-induced transcriptional
upregulation of CAPN1, CAPN2, IL‑6, TGFβ1,
angiopoietin 1 and collagen type Iα1274

MDL28170, calpeptin

Melanoma cell survival

Cisplatin-treated Me21
melanoma cells

Calpains• ↓ Activation of caspases 3 and 7• ↓ Cisplatin-induced apoptosis• ↑ Autophagy

160

MDL28170

Cardiacischaemia–reperfusion

Mice

CAPN1

• ↓ Spectrin or junctophilin 2 cleavage• ↓ Damage on mitochondrial function

117,
275

Chronic heart failure

Cardiomyocytes ofdogs of chronic heart
failureCalpains• ↓ Augmentation and altered kinetics of late
Na+ current

276

Leishmaniasis

In vitro growth ofpromastigotes ofL. amazonensis

L. amazonensis
calpain‡• ↑ Apoptosis (cell cycle arrest and DNA
fragmentation)

173

Chagas diseaseT. cruzi epimastigotes
infection in vitro

T. cruzi
calpain‡• ↓ Attachment to insect midgut• ↓ Differentiation• ↓ Viability

277

Pre-eclampsia

BeWo cells

Calpains• ↓ Apoptosis-inducing factor cleavage

278

Spinal cord injury

Rats with spinal cord
transection

Calpains• ↓ Cleavage of α‑subunit of voltage-gated Na+
1.6 channel• ↓ Persistent inward Na+ current• ↓ Spasms

249

MDL28170, mCalp‑I

LTP

Rats

CAPN1 andCAPN2

• ↓ LTP• ↓ SCOP degradation (MDL28170, but not
mCalp‑I)• ↓ PTEN degradation (mCalp‑I)

261

AK295, calpastatin
peptide (CAST)

Retinitis pigmentosa

C3H rd1/rd1 mice

Calpains• ↓ Photoreceptor cell death (AK295, short
term; CAST, short and long term)• ↑ Photoreceptor cell death (AK295, long term)

279

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Refs

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REVIEWS
Table 3 (cont.) | Selected in vitro and in vivo studies (since 2010) of calpain inhibition in disease
Inhibitor or vaccine

Disease/phenomenon

Model

Target*Effect

SNJ1945

Multiple sclerosis

EAE in mice

Calpains• ↓ Paralysis,• ↓ TH1 and TH17 cell inflammatory responses• ↓ Induction of calpain

22

Retinal ischaemia

Rats

Calpains• ↑ Electrophysiological function of innerretinal layers• ↓ Loss of cone‑ON bipolar and amacrine cells

280

Lissencephaly

Lis1+/− mice

Calpains• ↓ LIS1 cleavage• ↑ Corticogenesis,• ↑ Behavioural performance• ↑ Brain metabolism

17

Cardiacischaemia–reperfusion

Rats

Calpains• ↓ Spectrin-α and SERCA2A cleavage• ↑ Left ventricular function

281

Cortical impact
traumatic brain injury

Mice

Calpains• ↓ Spectrin-α cleavage

282

Machado–Joseph
diseaseLentiviral expressionof 72xGln‑ataxin 3 inmice

Calpains• ↓ Ataxin 3 cleavage• ↓ Cerebellar degeneration• ↓ Motor behavioural deficits

283

Ageing-related
syndromes

α‑Klotho−/− mice`

CAPN1• ↑ Reproductive ability• ↑ Body weight• ↓ Organ atrophy• ↓ Ectopic calcifications• ↓ Bone mineral density reduction• ↓ Pulmonary emphysema• ↓ Senile atrophy of skin133

Sickle cell disease

Hbbsingle/single SAD1 mice

CAPN1

• ↑ RBC morphology• ↓ RBC density• ↓ Hypoxia-induced RBC dehydration

220

Abdominal aortic
aneurysms andatherosclerosis

Hypercholesterolaemic CAPN1
Ldl−/− mice

• ↓ Spectrin‑α cleavage• ↓ Abdominal aortic width• ↓ Atherosclerotic lesion

26

CYLA

Retinal ischaemia

Rats

Calpains• ↑ Electrophysiological function of retina

218

A-705253 (BSF
419961)

Alzheimer disease

NMDAR-mediatedneurodegeneration
and Aβ‑induced
synaptic deficits inmice

Calpains• ↓ Neuronal cell death• ↓ Caspase 3 activation• ↓ Deficits in synaptic transmission

284

Alzheimer disease

Mice harbouring
APP:K670M or
APP:M671L,
presenilin 1:M146V and
tau:P301L mutations

Calpains• ↓ Tau hyperphosphorylation• ↓ Proteolytic cleavage of CDK5 subunit p35
to p2546

Alcoholism

Cue-induced
reinstatement of
alcohol-seeking
behaviour inpost-dependent
Wistar rats

Calpains• ↓ Cue-induced reinstatement of
alcohol-seeking behaviour• ↓ Alcohol (but not saccharine)-deprivation
effects• No significant induction of NMDAR-mediatedside effects (psychostimulant, cognition-impairing psychotomimetic effects)

285

Macrocyclic
aldehyde (CAT811)

Cataracts

Lambs with inherited
cortical cataracts

Calpains• ↓ Lens cytoskeletal protein cleavage• ↓ Cataract development

139

NH2-GRKKRRQRRR­
PPQPDALKSRTLRCOOH (Tat-μCL),
PD150606

Retinitis pigmentosa

Rats expressing
rhodopsin:S334ter

CAPN1• ↓ Retinal apoptosis

286

Dipeptidyl
α,β‑unsaturated ester

Malaria

P. falciparum
parasitaemia in humanerythrocytes or HeLa
cells

Possibly
Pf‑calpain• ↓ Parasite survival

287

BDA‑410

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Refs

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REVIEWS
Table 3 (cont.) | Selected in vitro and in vivo studies (since 2010) of calpain inhibition in disease
Inhibitor or vaccine

Disease/phenomenon

Model

Target*Effect

Refs

Hypervalentorganotellurium
compound (RF19)

Malaria

P. falciparum
parasitaemia in humanerythrocytes

Possibly
Pf‑calpain• ↓ Parasite survival

219

rSm‑p80(recombinant
protein)Schistosomiasis

Baboon or hamster

Smp‑157500(S. mansoni
calpain)• ↓ Adult worms and eggs in tissues (hamster)• ↓ Egg excretion in faeces and urine (baboon)• ↑ Protective immune response

10,
225

Aβ, amyloid-β; APP, amyloid precursor protein; CDK5, cyclin-dependent kinase 5; EAE, experimental autoimmune encephalomyelitis; IFN, interferon; IL, interleukin;
L. amazonensis, Leishmania amazonensis; LTP, long-term potentiation; MHC, major histocompatibility complex; MPTP, 1‑methyl‑4‑phenyl‑1,2,3,6‑tetrahydropyridine;
NMDAR, N-methyl-D-aspartate receptor; P. falciparum, Plasmodium falciparum; PTEN, phosphatase and tensin homologue; RBC, red blood cell; S. mansoni;
Schistosoma mansoni; SCOP, suprachiasmatic nucleus circadian oscillatory protein; SERCA2A, sarcoplasmic reticulum Ca2+ ATPase; TGF, transforming growth factor;
TH, T helper; TNF, tumour necrosis factor; Trypanosoma cruzi, T. cruzi. *No specific calpain was targeted in the study unless specified. ‡Involvement of other cysteine
peptidases has not been excluded.

inhibitor with good cell penetrability. Recently, a more
specific macrocyclic inhibitor, CAT811 (ointment form),
was tested on a sheep model of heritable cataract development; the inhibitor successfully slowed the development of the disease138,139 (BOX 1; TABLE 3; Supplementary
information S1 (figure)).
Calpain is also a therapeutic target for retinitis pigmentosa, which is caused by defects primarily in rhodopsinsbut also in various other genes, including those encoding
carbonic anhydrase, pre-mRNA processing factors andphosphodiesterases140 (TABLE 3). Retinitis pigmentosa ischaracterized by photoreceptor cell death, which resultsfrom calpain-mediated apoptosis induction141, increased
lysosomal membrane permeability 142 and/or downregulated heat shock protein 70 (HSP70)143. Thus, combining
calpain inhibitors with inhibitors of cathepsin and caspase,as well as HSP70 inducers, could be an effective strategy
for relieving the symptoms of retinitis pigmentosa.

Macrocyclic inhibitor
Macrocyclic structures
introduced into linear peptides
or compounds often improve
potency by providing
well-defined conformations,such as α‑helices and β‑strands
in the case of peptides, thereby
facilitating interactions with
their targets, which include
proteases. This strategy has
been used to develop
improved calpain inhibitors.

Cancer. The calpain–CAST system has demonstrated
opposing roles in cancer 30. In many cases, the upregulation of CAPN1, CAPN2 and/or CAPNS1 is observed,
whereas CAST is increased in others27,144–146. Activationof calpain-1 and calpain-2, and the proteolytic productsof their substrates, have critical roles in cancer cell survival. For example, MYC-nick, a calpain-mediated cleavage product of the MYC proto-oncoprotein, promotes
cytoskeletal remodelling, is upregulated in cancer cells
and facilitates cell growth under hypoxia and nutrient
deprivation, leading to malignancy 85,86. Calpain–CAST is
also involved in pathological angiogenesis, which is often
observed in tumours. This angiogenesis is promoted bya loss of CAST, which amplifies the activity of calpain-2(REF. 147). In addition, an accelerated calpain-mediatedcleavage of receptors, such as the androgen receptor 148
and human epidermal growth factor receptor HER2 (alsoknown as ERBB2)149, contributes to cellular resistance toanticancer therapies. Calpain-1 and calpain-2 also regulate cellular machineries for drug efflux and hence reduce
the efficacy of antitumour therapeutics such as tanespimycin, an HSP90 inhibitor derived from the antibiotic
geldanamycin29.
Recently, calpain-1 activity was shown to be importantin the treatment for myelodysplastic syndrome (MDS)150.
MDS is a haematopoietic stem cell disorder characterized

NATURE REVIEWS | DRUG DISCOVERY

by ineffective haematopoiesis and a tendency for acute
myeloid leukaemia progression. Lenalidomide, a synthetic glutamic-acid-derived immunomodulatory drug,
is used to treat MDS as well as multiple myeloma151. The
sensitivity of myeloid cells to lenalidomide is dependent
on calpain-1 but not calpain-2 activity, and suppressionof CAST increases the susceptibility of MDS to lenalidomide150. These results indicate that in the treatment of
MDS and possibly multiple myeloma, sufficient calpain-1
activity in the target myeloid cells is required for malignant cells to respond to treatment.
Unexpectedly, the unconventional CAPN3 (alsoknown as p94) and CAPN9 (also known as nCL-4) havealso been pathologically implicated in cancers. For example, CAPN3 is highly expressed in melanoma cell lines152
and is downregulated in response to interferon-γ treatment 153. CAPN3 is also found in bovine bladders withurothelial tumours154. Conversely, the CAPN3 isoforms
hMp84 and hMp78 are upregulated in human melanoma
cell lines treated with cisplatin to induce apoptosis, anddownregulated in biopsies from human malignant melanocytic lesions144. Consistent with these observations,overexpressing hMp84, but not inactive hMp84:C42S, inmelanoma results in cell death155.
CAPN8 (also known as nCL-2) and CAPN9 are predominantly expressed in the gastrointestinal tract, andCAPN9 but not CAPN8 is suggested to be involved in
the suppression of tumorigenesis156–158. Recently, downregulation of human CAPN9 but not CAPN8 was shownto correlate with unfavourable prognosis in patients withgastric cancer 159. An expression study using stable humangastric cancer cell lines (MGC80‑3 and MKN‑45) showedthat CAPN9 but not CAPN8 induced G1 cell cycle arrest
and caspase-mediated apoptosis159.
Other studies have suggested that calpain is required for
apoptosis induced by the anticancer drug cisplatin in various cancer cell models144,160, and that calpain inhibition isboth beneficial and detrimental depending on the stage oftumour progression145. Thus, further studies are required
to clarify whether calpains can both promote and suppress
tumour growth and metastasis in different settings. Moresystematic studies examining, for example, whether the
involvement of calpains depends on the cell type and/or
calpain species, should clarify whether calpain-targeted
strategies would be beneficial in cancer therapies.
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Calpainopathies
LGMD2A, the first calpainopathy. An importantfunction of calpains was confirmed when CAPN3 was
identified as the responsible agent for LGMD2A32,93,161.
Prompted by this groundbreaking discovery, the term
calpainopathy was explicitly defined as a disease condition that results from mutations in calpain genes. CAPN3
is predominantly expressed in skeletal muscle and was
the first non-ubiquitous member of the calpain superfamily to be reported60. LGMDs are characterized by theirselective effect on the proximal muscles of limb girdles162(see also the Calpain 3 page of the Leiden Muscular
Dystrophy website; see Further information). Worldwide,
LGMD2A, an autosomal recessive LGMD, is the mostfrequent form of LGMD, and the rate of LGMD2A occurrence in some regions is much higher than average162.
In LGMD2A, CAPN3 is devoid of protease function93.
This finding is in stark contrast to the general conceptthat the pathology of muscular dystrophies is aggravated by a secondary overactivation of conventional calpains130. Moreover, studies using various mouse models
of LGMD2A have revealed that CAPN3 also has a role asa regulatory component for Ca2+ release from sarcoplasmic reticulum in muscle cells in a manner independentof its protease activity 163,164. Therefore, inhibiting CAPN3
would not be effective for LGMD2A and in fact could be
detrimental (for an exception, see below).
Accordingly, the objectives of CAPN3‑targeted therapy for LGMD2A are to restore or compensate for theloss of CAPN3 function53. To achieve these goals, essential questions that remain to be answered include how
CAPN3 functions both as a protease and as a non-proteolytic modulator in muscle cells, what the in vivo targetsof CAPN3 activity are, and in which biological pathways
CAPN3 plays a part.

Neglected tropical diseases
Among various parasitic andinfectious diseases, 17
diseases have been recognizedby the World HealthOrganization as targets forwhich control would promote
an exodus from poverty
somewhere in the world.

Other calpainopathies. CAPN5 is responsible for autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV)34,71. CAPN5 is expressed in mosttissues, especially in the central nervous system165,166.
So far, R243L, L244P and K250N have been identified
as ADNIV-causative CAPN5 mutations. Among these,
R243L appears to be hyperactive, and transgenic miceoverexpressing CAPN5:R243L show an ADNIV-likephenotype167, whereas Capn5−/− mice exhibit no apparent
phenotype165. Therefore, inhibiting CAPN5 (also known
as hTRA3) in the retina is a possible therapeutic strategy for ADNIV; however, whether the Ca2+-dependent
proteolytic activity of CAPN5 is similar to those of conventional calpains needs to be explored. Alternatively, amutant CAPN5‑specific siRNA may be considered as atherapeutic approach.
CAPN14 has been identified as a genomic locus thatis specifically associated with another calpainopathy, theallergic disorder eosinophilic oesophagitis35,36. CAPN14 is
predominately expressed in the oesophagus and is upregulated in eosinophilic oesophagitis or by IL‑13 stimulation35. Intriguingly, an eosinophilic oesophagitis-associatedrisk single-nucleotide polymorphism (SNP) in CAPN14
is intronic and decreases the expression level of CAPN14.
The characterization of the protease activity of CAPN14

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and its possible substrates suggests that downregulationof CAPN14 compromises the cellular responses to IL‑13
signals, whereas excess CAPN14 activity impairs epithelial
barrier function168. The restoration of CAPN14 may therefore represent a logical therapeutic strategy, but its potential cytotoxicity must be monitored. Intriguingly, although
the human CAPN14 gene is localized to a genomic region
that is syntenically conserved in mice and rats, mouse or
rat Capn14 cannot be found. This finding suggests that
another calpain species in some rodents assumes the functions of CAPN14 and/or that the calpain-related aspect of
the eosinophilic oesophagitis pathology does not present
in these rodents.
More recently, another calpainopathy was reported:
spastic paraplegia 76, a neurological disorder linked
to CAPN1 (REF. 48). Pathogenic mutations in CAPN1
and other experimental evidence indicate that spastic
paraplegia 76 is caused by a loss of calpain-1 function.
Although no gross neurological abnormality is apparent
in the constitutive Capn1−/− mouse, calpain-1 is known tohave a neuroprotective role and to contribute to synaptic
plasticity 18,169. In addition, missense mutations in CAPN1
are associated with spinocerebellar ataxia in dogs170 andhumans171. Taking this evidence together, it is strongly
anticipated that calpain-1 will be a target not only forinhibition but also for genetic restoration therapeuticstrategies.
Finally, with regard to additional potential calpainopathies, functional effects of SNPs in CAPN8 and CAPN9
merit attention. CAPN8 and CAPN9 are expressed in thegastrointestinal tract, especially in the mucus-secreting
cells of the stomach, where they function as a heterodimer in complex with each other (called calpain-8/9 or
G‑calpain (G for gastric))33. Capn8−/−, Capn9−/− andCapn8C105S/C105S mice are significantly more susceptible
to gastric ulcers induced by ethanol stress than wild-type
mice33. Importantly, several SNPs that inactivate CAPN8
or CAPN9 exist, suggesting that a susceptibility to gastriculcers can be predicted by these SNPs. Therefore, strategies that compensate for and/or activate calpain-8/9,
depending on the effect of the SNP, would be logicaldirections for treatment.

Infectious diseases
Parasitic diseases. Calpains expressed in parasites play
crucial parts in their pathogenicity; thus, calpains represent promising anti-disease targets. Notably, calpains are
involved in the pathogenicity of trypanosomiasis172, leishmaniasis172,173 and schistosomiasis174, which are among theneglected tropical diseases recognized by the World HealthOrganization175.
Malaria caused by Plasmodium falciparum is one of
the most serious parasitic diseases176. P. falciparum uses
aspartyl and cysteine proteases to degrade host haemoglobins and to survive as a parasite177, and Pf‑calpain isessential for the life cycle of the parasite in cells178, suggesting that these proteases are promising drug targets.
Although host calpain-1 is reported to be required forthe efficient egress of P. falciparum41, the invasion andgrowth of parasites in erythrocytes from wild-type andCapn1−/− mice show no significant difference179.
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Table 4 | Selected calpain-related therapeutic agents
Compound(alternative names)

Company or instituteDiseasePhase

Details

Refs

ABT‑957

AbbVieAlzheimer disease

Phase I,
terminatedRandomized, multiple-dose-escalation safety,
tolerability and pharmacokinetics study288

ABT‑957

AbbVieAlzheimer disease

Phase I,
terminatedRandomized, multiple-dose-escalation safety
and efficacy study

289

Olesoxime

Trophos

Relapsing-remitting Phase I,
multiple sclerosis
completed

Randomized, single-dose safety study

290

Olesoxime

F. Hoffmann‑La Roche

Spinal muscularatrophyOlesoxime

Trophos

Amyotrophic lateral Phase II/III,sclerosis
completed

Multicentre, open-label safety extension study

Cyclosporine A

E. HallTraumatic brain
injuryPhase I/II, in
progress

Randomized, pharmacokinetics andpharmacodynamics study; evaluated bycalpain-mediated cytoskeletal breakdown
products

E-64d (EST, Estate,loxistatin andrexostatine)

Taisho Pharmaceutical

Muscular dystrophy

Phase III,
Effective for protease inhibition to delay
discontinued muscle protein degradation and diseaseprogression45

AK‑295 (CX295)

Alkermes• Stroke• Cataract

Preclinical,
Systemic inhibition of calpain to suppress side
discontinued effects of cancer therapy on sensory neurons

295

C-101 (myodur and
CLA)

CepTorMuscular dystrophy

Preclinical,
Increased myofibre diameter in mdx mice anddiscontinued low toxicity for rats

127

CYLA

The State University of
New York• Multiple sclerosis• Retinal ischaemia

Preclinical

Capable of crossing the blood–brain barrier
through taurine transporter and effective forretinal ischaemia-associated lesion

217,218

C-201 (neurodur and
CLA)

CepTor• EAE• Acoustic trauma

Preclinical

Aldehyde form of CYLA; effective for slowing
EAE progression in mice and treating acoustic
trauma in chinchillas

214

CEP‑3453

University of Pennsylvania, Ischaemic stroke
Teva

Preclinical

Neuroprotective 22 h post-ischaemia
treatment in a rat model

296

CEP‑4143

Cephalon

Spinal cord injuries

Preclinical,
Pre-injury treatment exhibited
discontinued neuroprotection in rat model

297

Calpeptin (IPSI‑001)

Medical University ofSouth Carolina

Multiple sclerosis

Preclinical

Reduction of demyelination and axonal
damage to treat multiple sclerosis-associatedoptic neuritis

272

Calpeptin (IPSI‑001)

Washington University
School of Medicine

Wolfram syndrome

Preclinical

One of disease-responsible gene products,
WFS2, downregulates CAPNS1 via theubiquitin–proteasome system

298

A-705239 (BSF
409425)

Abbott• Acute myocardial
infarction• Cerebrovasculardiseases

Preclinical

Respiratory functions of mitochondria
from the heart challenged by ischaemia–
reperfusion was preserved

A-705253 (CAL 9961
and BSF 419961)

Christian-Albrechts
University of Kiel

Acute myocardial
infarction

PreclinicalPre-infarction administration inhibited calpain
and cardiac hypertrophy299

Ala‑1.0 (leupeptin
linked to pregabalin)City University of
New York, Instituteof Basic Researchin Developmental
Disabilities, The State
University of New York• Stroke• Alzheimer disease• Muscular
dystrophy

PreclinicalIntraperitoneal administration immediately
after controlled cortical impact decreased
neurodegeneration in a rat model216

rAAV2/1‑CAPN3

French National Center
for Scientific Research(CNRS)

Muscular dystrophy

PreclinicalIntramuscular delivery of CAPN3 genecorrected dystrophic features of LGMD2A
model mice300

• Sm‑p80‑pcDNA3• Sm‑p80‑VR1020

Texas Tech University
Health Sciences Center

Schistosomiasis

PreclinicalLongevity of production of Sm‑p80-specificantibodies (1–8 years) was demonstrated

226,301

rSm‑p80

Texas Tech University
Health Sciences Center

Schistosomiasis

PreclinicalEffect of vaccination to acute and chronic
infection was shown

Phase II/III, in Single-dose safety, tolerability and efficacy
progressstudy

291
292,293294

199,200

225

EAE, experimental autoimmune encephalomyelitis; LGMD2A, limb-girdle muscular dystrophy type 2A.

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Another parasite spread by insects is Trypanosoma,
which causes African sleeping sickness (Trypanosoma bru‑
cei transmitted by Glossina spp. (tsetse fly)), Chagas disease(Trypanosoma cruzi transmitted by Reduviidae spp. (assassin fly)), kala-azar and Oriental sore (Leishmania dono‑
vani and Leishmania major transmitted by Phlebotominae
spp. (sand fly)), as well as other diseases172. Surprisingly,
these parasites have 18–27 calpain genes42, one of which,
ClpGM6, is involved in the morphology determinationof T. brucei 180. ClpGM6 is an 820 kDa protein containing
three copies of the CysPc and CBSW domains, and >70
repeats of the GM6 motif (a 68 amino acid residue unit)(FIG. 1). It is still unknown whether trypanosomal calpains
function as proteases because most of them lack the conserved active site cysteine residue42.
The blood flukes Schistosoma mansoni, Schistosoma
japonicum and Schistosoma haematobium are transmitted by Planorbidae spp. (planorbid or ramshorn
snails) and cause schistosomiasis, which can include
hepatosplenic inflammation, liver fibrosis and bladdercancer 174. S. mansoni harbours seven genes that encode
calpains, four of which are classical calpains and one
is a CAPN7 homologue. At least one of the classicalcalpains, Smp‑157500 (FIG. 1), may be essential for thepathogenicity of this parasite given that it is an effective
target for vaccines10 (TABLES 3,4 and see below). The
physiological functions of these calpains, however, arestill elusive50.
Fungal and bacterial infections. Of the 449 whole-genome-sequenced fungus genera, 431 have one or more
calpain-encoding genes; important exceptions include
Schizosaccharomyces, Encephalitozoon and Pneumocystis(see MycoCosm of the US Department of Energy’s Joint
Genome Institute Genome Portal; Further information).
Candidiasis, cryptococcosis, aspergillosis, coccidioidomycosis and trichophytia are caused by infection withthe fungi or yeasts Candida spp., Cryptococcus spp.,
Aspergillus spp., Coccidioides spp. and Trichophyton spp.,
respectively, of various human tissues, such as the lung,mouth and skin43. These infections cause serious problems, especially in immunocompromised individuals,
such as HIV-infected or organ transplant patients.
To infect human tissues, the pathogens describedabove adapt to the environmental pH by a mechanismthat uses their own calpains, PalB/Rim13 (REFS 181,182).
RIM13 is the only Saccharomyces cerevisiae gene that
encodes a calpain183, and palB is one of the calpain genesof Aspergillus nidulans 184; they are both orthologues ofhuman CAPN7. The involvement of Rim13 and PalB inalkaline adaptation signalling was discovered in A. nid‑ulans and S. cerevisiae, followed by elucidation of thedetailed process54. A pH sensor (consisting of Rim21/
Dfg16/PalH (7 spanners), Rim8/PalF (arrestin-like) andRim9/PalI (chaperone)) transduces an alkaline signal toa proteolytic complex with the aid of ESCRT (endosomal sorting complex required for transport)-I, II and
III. In this process, Rim20/PalA and Ygr122w/PalC are
induced to act as a scaffold for the proteolytic activationof Rim101/PacC (transcription factor) by Rim13/PalB54.
Thus, this signalling is called the Rim101 pathway.
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Among the few bacterial calpains 52 , Tpr ofPorphyromonas gingivalis, which causes periodontitis,
is required for the survival and infection ability of thebacteria, and was shown to be a Ca2+-dependent cysteine
protease with both autolytic and substrate (for example,
fibrinogen) proteolysing activities12.

Strategies for therapeutics
As discussed above, there are three types of calpain-related
human disorders: those exacerbated by human calpain
activities (type 1); those caused by pathogenic microorganisms that use the host’s and/or their own calpains for
infection and survival (type 2); and those caused by calpain
gene deficiencies (type 3). For most of the type 1 and someof the type 2 disorders, inhibitors for the conventional calpains are the first therapeutic choice. Some microorganisms
in type 2 disorders can infect or survive using their own calpains, which may therefore represent ideal specific targets.
For type 3 disorders with defective calpains, gene therapy torestore calpain activity should be considered as a potential
therapeutic approach. In addition, some type 3 disorders
result in hyperactivation of calpains due to a gain‑of‑function mutation167, for which the inhibition or reduction of
calpain activity would be a logical therapeutic strategy.
Inhibiting conventional calpain activities. Firstgeneration calpain inhibitors include leupeptin and E-64,
originating from actinomycete and fungus, respectively,
and their derivatives. These inhibitors exhibit little specificity for calpains and show broad inhibitory activity
against not only clan CA proteases118 but also matrix metalloproteinase 2 (REF. 185) (BOX 1; TABLE 3; Supplementary
information S1,S2 (figure, table)). Nevertheless, leupeptin
and its modified versions have been studied extensively
and evaluated in pilot clinical trials186. One of these products, C-101 (also known as myodur; see below), although
discontinued as a clinical candidate, represents an important approach to effectively deliver a drug to muscle cellsby taking advantage of a receptor–ligand interaction127(see below) (Supplementary information S1 (figure)).
Meanwhile, E-64d was shown to prolong the lifespan of adystrophic hamster, UM‑X7, and was used in clinical trials
up to phase III in the 1980s45. Unfortunately, the results
were inconclusive, and the trials were discontinued in
1992 (REF. 187). Several attempts to improve the specificityof these molecules resulted in second-generation inhibitors such as SJA6017 (REF. 92) and PD150606 (REF. 76).
However, these agents were still not sufficiently specificto distinguish calpains from other proteases. Althoughendogenous CAST is currently the only absolutely specific inhibitor for classical calpains, strategies to improve
inhibition are emerging (BOX 1).
Indeed, advances in structural biology have enabled
logical structure design, which has led to a group ofpromising third-generation inhibitors with fixed secondary structures. Although proteases are known togenerally recognize β-strands188, most of the small-molecule protease inhibitors are conformationally flexible.
This flexibility induces cooperative effects between theprotease and the inhibitor (so‑called induced fit 189),
resulting in unexpected enzyme–inhibitor interactions
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Intrinsically unstructured
proteinA protein that does notpossess a fixed or stable 3D
structure (also called an‘intrinsically disordered
protein’). Some of these
proteins remain unstructured
even in their functional state,
whereas others adopt a fixed
structure after binding toanother protein. For example,
calpastatin has tandemly
repeated calpain inhibitory
sequences, neither of whichassumes a defined structure
unless calpastatin is bound to
calpain.

at adjacent or even remote locations from the active siteand, therefore, a loss of inhibitory specificity. This effectcan be avoided by restricting the secondary structureof the inhibitor molecule. Among several attempts toachieve this goal, stable β‑strands were achieved by themacrocyclization of inhibitor peptides138,190,191.
Notably, although CAST is an intrinsically unstructured
protein, the 3D structure of a calpain-2–CAST complex
shows that α‑helix and β‑turn structures in CAST subdomain B induced by its binding to the active site of calpain-2 have essential roles in its specific inhibition. Thus,
peptides made to mimic these structures by crosslinkers192 or by cyclization188 are inherently specific to calpains (with inhibition constant (Ki) values of 10–20 μM)(Supplementary information S1 (figure)).
In addition, an alternative approach is provided by acalpain-derived inhibitory peptide for mitochondrial calpain-1 that specifically blocks calpains193. Mitochondrial
calpain-1 is associated with the molecular chaperone
ERp57 via the CBSW domain of CAPN1 (REF. 194).
Because inhibiting ERp57 destabilizes mitochondrial
calpain-1 (REF. 194), synthetic peptides were screened fortheir ability to competitively inhibit the ERp57–CBSW
interaction. This screen identified PDALKSRTLR, anoligopeptide corresponding to a linker region between
the PC2 and CBSW domains of CAPN1 that inhibited
mitochondrial but not cytosolic calpain-1 (medianinhibitory concentration (IC50) of 112 nM)194. A form of
this peptide N‑terminally conjugated with the cell-penetrating HIV‑1 Tat sequence (GRKKRRQRRRPPQ)
prevented photoreceptor cell death in retinal dystrophic
rats (TABLE 3), with no inhibition of cathepsin L, papain
or proteasomes194. This strategy is ideal in its specificity,and may be applicable to other calpains: for example,
for the development of a peptide that interferes with the
PEF(L) and PEF(S) interaction of conventional calpains.
As described above, the development of allostericinhibitors of calpains is another important area for
improving specificity. For this endeavour, elucidation of
the precise inhibitory mechanisms of α‑mercaptoacrylicacid derivatives, such as PD150606, is urgently required.
Intriguingly, the dimerization of PD150606 and its
derivatives via disulfide formation significantly increases
their calpain inhibitory activity (for example, the IC50
values of PD150606 and its dimer are 5.0 μM and 7.5 nM,
respectively)195. The dimerized molecules bind to the
PEF domain similarly to PD150606 but more stably 195,
although their inhibitory mechanisms are unclear. Thesestudies have opened up a new direction for calpain-specific inhibitor development, in which the inhibitors are
directed to regions other than the active site.
As a promising example, a novel allosteric site and its
small-molecule inhibitor, NSC13345, for cathepsin K, acysteine cathepsin with 3D structural similarity to thecalpain CysPc domain, were discovered by computational methods196. NSC13345 inhibits cathepsin K bybinding to a relatively flat side surface of the β‑strandrich region that harbours the active site histidine andasparagine residues, corresponding to the PC2 domainof calpains196. Therefore, similar methods should beapplicable to finding calpain-specific allosteric sites and

NATURE REVIEWS | DRUG DISCOVERY

their docking molecules. Further information about the
recent progress on calpain inhibitors can be found inother detailed and comprehensive reviews3,197,198.
Preclinical and clinical agents in development. Calpain
inhibitors are continuing to be developed therapeutically,and some of them are currently being tested in clinical trials (TABLE 4). Two structurally related α-ketoamide calpain
inhibitors, A-705239 (also known as BSF 409425) andA-705253, show promise because of their improved watersolubility, cell permeability and metabolic stability overother inhibitors, although they have some limitations
with regard to specificity (Supplementary information
S1,S2 (figure, table))199. Administering A-705239 after an
induced traumatic brain injury in rats rescued brain cells,
proving the biological efficacy of the drug 199. In anotherdisease model, acute myocardial infarction inducedby ischaemia–reperfusion in a rabbit heart, the infarct
size was reduced and the respiratory function of mitochondria was preserved when A-705239 was included
throughout the procedure200. An improved derivative of
A-705239 developed by AbbVie, ABT‑957, had been in
the first sets of clinical trials as a treatment for cognitive
disorders until recently 198,201–203 (TABLE 4). The exact structure of ABT‑957 has not yet been disclosed.
A neuroprotective cholesterol derivative, olesoxime(Supplementary information S1 (figure)), which has been
subjected to clinical trials as a therapeutic reagent for
motor neuron diseases such as ALS204, spinal muscularatrophy (SMA)205,206 and relapsing-remitting multiple sclerosis207, was recently shown to suppress calpain activity ina rat model of Huntington disease208,209. The precise modeof action of olesoxime is currently elusive, but as it binds
to outer mitochondrial membrane proteins and inhibits
the efflux of apoptotic factors under neurotoxic or cytotoxic conditions in vitro210, calpains are probably not its
direct target. The safety and suitability of olesoxime fororal delivery have been demonstrated in clinical trials206,211(TABLE 4). A beneficial clinical outcome, such as a retardation of disease progression, was observed in a SMA trial
focusing on the early phase of disease onset205, but not in
an ALS trial assessing survival at the end stage of the disease211. These results suggest that the neuroprotective effectof olesoxime depends on the timing of its administration
during disease progression. Although the calpain activity
in olesoxime-treated neurodegenerative diseases otherthan Huntington disease awaits investigation, it is possible that one of the main neuroprotective actions of olesoxime depends on calpain suppression. Notably, survival of
motor neuron (SMN) protein, a gene product responsible
for SMA, is a calpain substrate212, and calpain upregulation
has been reported in an ALS mouse model213, suggestingthat other calpain inhibitors may also be effective for treating these diseases as well as Huntington disease.
In parallel with the efforts to refine the core structure of calpain inhibitors, the covalent attachment ofa tag motif to an inhibitor molecule that causes it toaccumulate in a region of interest, such as muscle cells,
has been examined127,214–216. Among these compounds,
C-101 is a modified leupeptin linked to carnitine, whichis efficiently targeted to skeletal muscle tissue, where the
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REVIEWS
carnitine receptor OCTN2 (also known as SLC22A5) isexpressed127. At the preclinical level, C-101 increased
the fibre diameter in the skeletal muscle of mdx mice,indicating its promise as a therapeutic reagent for muscular dystrophies127. However, the preclinical trial forthis product was discontinued, and other variants thathave been examined at the preclinical level await further
evaluation. These inhibitors include C-201 (also known
as neurodur)214, GABAdur 215 and Ala‑1.0 (REF. 216),
which are attached to taurine or the anti-epileptic drug
pregabalin and are designed to elicit neuroprotection
by limiting calpain activation (Supplementary information S1 (figure)). CYLA is a diethyl acetal of C-201 that isconverted to an active form after undergoing hydrolysis
in vivo. Delivery of CYLA to the brain and prevention ofaxon injury was demonstrated in a mouse model of multiple sclerosis217. CYLA is also reported to prevent retinal
cell degeneration in a rat model of retinal ischaemia218.

Targeting calpain pathways in infectious diseases
Malaria and sickle cell disease. As the malaria-causing Plasmodium parasite requires proteases, including
Pf‑calpain, for its survival, these proteases are promising
drug targets. Conventional calpain inhibitors also inhibit
Pf‑calpain: ALLNal and ALLMal suppress the erythrocyte invasion of P. falciparum40, and BDA‑410 blocks parasite growth in vitro and in vivo9. In addition, hypervalentorganotellurium compounds inhibit the Pf‑calpain-like
activity 219 (TABLE 3). These potential drug candidates
should be tested for their specificity using recombinantPf-calpain70.
Sickle cells disease (SCD) is caused by pathogenic
mutations of the β‑globin gene (HBB) and is accompanied by malaria resistance. Dense sickle cells are ahallmark of human SCD and show reduced levels of
CAST220. Notably, the oral administration of BDA‑410
in a mouse model of SCD (SAD mice) ameliorates sickle
cell density and hypoxia-induced erythrocyte dehydration220. The proposed molecular mechanisms of sickle
cell deficiency include the perturbation of PRX2 (alsoknown as calpromotin), which protects against oxidative stress, and the upregulation of a Ca2+-activated
K+ channel (Gardos channel) by overactivated calpain-1(REF. 220). Consistent with this scenario, Gardos channel
activity is suppressed in Capn1−/− mouse erythrocytes221.
However, the Capn1−/− mouse erythrocytes become
deformed upon Ca2+-ionophore-induced echinocyte
formation221. Thus, further investigations of the effectsof calpain-1 inhibition on erythrocytes need to be conducted before applying calpain inhibitors to treat SCD.
Trypanosomiasis and leishmaniasis. MDL28170 shows
trypanocidal effects without significant toxicity to thehost cells173 (TABLE 3). Treating Leishmania amazonen‑sis promastigotes with 30 μM (double the IC50 dose)of MDL28170 efficiently suppresses their growth andviability, and induces an apoptosis-like cell morphology
accompanied by cell cycle arrest and DNA fragmentation173. ALLNal, however, suppresses the apoptosis-likecell death of L. donovani induced by miltefosine (anothertrypanocidal agent), and E-64 has no effect 222. These
16 | ADVANCE ONLINE PUBLICATION

studies collectively suggest that the target of MDL28170
is unlikely to be calpain activity, but that Trypanosomal
calpain-like molecules, such as CAP5.5 (TbCALP1;
FIG. 1), that lack protease activity may act as cytoskeletal
modulators180,223. Although there is evidence to suggestthat trypanosomal calpains would be good drug targets,
inhibitors such as MDL28170 also act on host calpains,and detailed studies are needed to determine how thepseudo-proteolytic trypanosomal calpains function andhow calpain inhibitors act against them.
Schistosomiasis. There is currently no vaccine for schistosomiasis for human use. However, one of the leading
candidate target molecules for a schistosomiasis vaccine is Sm‑p80, a C‑terminal portion of the S. mansoni
calpain Smp‑157500 (FIG. 1). Smp‑157500 is exposed on
the membrane surface and has an important role in thesurface membrane renewal and recycling of the parasite to evade the host immune response10,224. Although
the precise function of Smp‑157500 is currently elusive,
baboon vaccination data for Sm‑p80 are promising 10,225(TABLES 3,4). Baboons chronically infected with S. man‑soni were treated with a recombinant Sm‑p80 protein(rSm‑p80) or an Sm‑p80‑expression DNA vector plasmidalong with adjuvants (Toll-like receptor 4 agonist-based
glucopyranosyl lipid (GLA‑SE) or aluminium hydroxide(alum))10. Among several combinations, rSm‑p80 plus
GLA‑SE was the most effective, resulting in the production of immunoglobulin A (IgA) in addition to IgG and
IgM, a 36% reduction in the number of worms, and 54%
and 33% reductions in the amounts of tissue and faecal
eggs, respectively 10. The same strategy was also effective
in baboons and hamsters infected with S. haematobium,
the Sm‑p80 amino acid sequence of which is 95% identical to that of S. mansoni 225. Surprisingly, the elicited
Sm‑p80‑specific IgG in vaccinated baboons was stilldetected 5–8 years after immunization226. These studies support the testing of an Sm‑p80 vaccine in humanclinical trials.
Diseases caused by fungi, yeasts and bacteria. Inhibitors
specific for calpains in fungi, yeast and bacteria are promising therapeutics for diseases caused by these pathogens.
For example, C. albicans infects and lives on mucosal
surfaces of the human gastrointestinal and genitourinary
tracts by expressing several genes for alkaline adaptation, such as superoxide dismutase 4 (SOD4) and SOD5
and aspartyl proteases (SAP5 and SAP6), causing candidiasis13. Deletion of RIM101 effectively disrupted thisinfection by downregulating Rim101‑induced genes13.
Similarly, Cryptococcus neoformans uses the Rim101 pathway for infection, and causes life-threatening meningitis in
immunocompromised humans182. Rim13 proteins of theseyeasts are homologues of PalB and CAPN7 (also known
as PalBH), which constitute the most divergent calpain
subfamily, and have further diverged CysPc domains evencompared with that of human CAPN7 (~20% identity)50.
Thus, it is possible to design inhibitors specific to theseyeast Rim13 proteins without inhibitory activity to human
CAPN7, which is thought to be essential for human cellular functions227. However, so far, no inhibitor has beenwww.nature.com/nrd

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Box 2 | Activators of calpains
Many enzymes have efficient activators, such as phorbol esters and diacylglycerol for
protein kinase C, AMP for AMPK, and the small GTPase RAC for NADPH oxygenase.
Calpain activators could be used, for example, to increase calpain activity in attenuatedactivity-type calpainopathies and in some cardiovascular disorders requiring a transient
activation of calpains. The conventional calpains are activated by Ca2+, Mg2+ andphospholipids, which are common activators for various enzymes, and hence could not
be used as a specific activator. Although activator macromolecules for calpains (UK114,
acyl CoA-binding protein, DNA and calpastatin fragments, among others) have beenreported, none of them has survived further analysis. Why is a calpain activator so
elusive?
One possible reason is that calpain activity has to be strictly suppressed in the cell. In
fact, conventional calpains proteolyse more than 40% of the peptide bonds of mostpolypeptides when exhaustively reacted in vitro (F. Shinkai-Ouchi, Y.O., T.C.S. and H.S.,
unpublished observations). Thus, there are multiple safety features that regulate theactivity of conventional calpains, such as the very high [Ca2+] requirement for full
activity and the lack of an active site conformation in the absence of Ca2+. In addition,
the deep active site cleft is inaccessible for many structured polypeptides, and thespecific inhibitor calpastatin is expressed in excess amounts in most cells. However, therecently reported intermolecular complementation phenomenon of CAPN3 (REF. 232)
provides a potential new direction for the development of a calpain activator, in thatsome parts of calpain itself might function as an activator.

designed, and no 3D structures have been determined for
Rim13. These topics are challenging but urgent for future
study. Conventional calpain inhibitors may also be effective in controlling these diseases. For example, Shigella flex‑
neri, a dysentery-causing bacteria, does not have its own
calpain but escapes from the host immunity by activating
conventional calpains228.

Intermolecularcomplementation(iMOC). A phenomenon inwhich single-polypeptide-de‑
rived fragments, none of which
are capable of expressing theactivity of the original protein,reconstitute the originalactivity through spontaneousand noncovalent interaction
under physiological conditions.
In this process, the amino acids
essential for the activity areprovided by different
fragments.

Restoring intrinsic functions of calpains
LGMD2A is the most thoroughly studied human calpainopathy confirmed to be caused by the genetic
loss‑of‑function of a calpain gene (CAPN3), and is therefore the leading example for which the complementation of calpain activity would be an appropriate therapy.
Among other calpainopathies reported so far, thosecaused by defects in CAPN1 (REF. 48), CAPN8 (REF. 33),
CAPN9 (REF. 33) and CAPN14 (REFS 35,36) would also
require restoration by complementation.
There are several challenges facing the diagnosis
and treatment of LGMD2A. A definitive diagnosis of
LGMD2A requires the identification of pathogenic
mutations in CAPN3, which encompasses more than
60 kb, and this test is time consuming and costly. A frequently used alternative is to check the CAPN3 protein
by western blot analysis. However, a normal level of
CAPN3 is found in ~30% of patients with LGMD2A162,
and clinicians need to be aware of this fact when using
this method. Analysing the Na+-dependent autolytic
activity, a unique feature of CAPN3 (REF. 229), to assess
the activity of CAPN3 would be a better diagnostic
method. Furthermore, the nature of the protease activityof CAPN3 remains unclear: that is, where it is activated,
in what forms and under what circumstances.
Gene therapy. Gene therapy is currently the most practical
approach for calpain replacement because the sizes of calpains (~100 kDa) are appropriate for viral vectors, and noactivators for these calpains are currently available (BOX 2).

NATURE REVIEWS | DRUG DISCOVERY

In the course of developing strategies for correcting
genetically defective CAPN3 by gene transfer, it was
revealed that skeletal but not heart muscle has the capacity to regulate CAPN3, and that freely active CAPN3
can be detrimental to tissues in which it is accidently
expressed47,59. To address this issue, skeletal-muscle-specific promoters and/or cardiac-specific gene suppression by microRNAs are effective strategies that do notdecrease the expression level of the transferred CAPN3
gene in skeletal muscle47 (TABLE 4).
Therapeutic strategies to correct causative gene mutations by gene editing techniques combined with the use ofpatient-derived induced pluripotent stem cells are also on
the horizon230,231. Therapies for calpainopathies, depending on the nature of the affected tissues and the mutation,
could greatly benefit from such advancements. More than
one-third of the cases of LGMD2A are caused by missense mutations162 (see also Further information), which
are appropriate for gene editing therapy.
Intermolecular complementation of CAPN3. An alternative approach to correct CAPN3 activity is suggested
by an unusual activation ability of CAPN3. CAPN3
undergoes rapid autolysis; however, it was recently discovered that the protease activity, including its autolytic
activity, can be restored through the intermolecular com‑plementation (iMOC) of autolysed fragments232. Such
iMOC had only previously been described for some
proteases of viruses, the self-remodelling of which is
involved in efficient infectivity 233. The iMOC of CAPN3
is thought to be a mechanism for regulating its activity
and localization232. Therefore, in some specific cases,
it might be possible to correct dysfunctional CAPN3
mutants by delivering a defined region of CAPN3,
which is not in itself active because it is not the whole
molecule232,234.
Inhibition of LGMD2A mutant CAPN3. An in vitro
study indicated that some LGMD2A mutant CAPN3
proteins, such as those mutated in the PEF domain,
have accelerated autolytic activity, so they cannot function owing to their rapid autodegradation93,235. It may
therefore be possible to use partial inhibitors that slow
down this autolysis but do not competitively inhibit theprotease activity. For example, PD150606 binds to PEF
domains76, although its primary site of action is the CysPc
not the PEF domain (see above)79. One of the amino acidresidues, F226 of PEF(S), that contacts PD150606 or
its derivatives, is conserved in the PEF(L) of CAPN3, aregion of LGMD2A pathogenic mutation (F779I)236 (seealso Further information). Therefore, PD150606 and/or its derivatives may alter the activity of PEF-domainmutated CAPN3 proteins with moderate efficiency. In
addition to LGMD2A caused by hyper-autodegrading
CAPN3 mutations, some other conditions may be ameliorated by inhibiting CAPN3. For example, tibial muscular dystrophy is primarily caused by mutations in the titin(TTN) gene, which in turn dysregulate CAPN3 (REF. 237).
Cardiomyopathy phenotypes collaterally caused by misexpression of CAPN3 in heart muscle, which is a problem
in applying gene therapy for LGMD2A47, could also be
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REVIEWS
prevented by inhibiting CAPN3 in the heart (TABLE 4). In
these cases, the development of CAPN3‑specific inhibitors would be helpful.
Increasing muscle mass. A different approach to treat
LGMD2A, as well as other muscular dystrophies, is topromote muscle development and regeneration, as is
the aim of anti-myostatin treatment238,239. In this respect,
CAPN6 (also known as CANPX), the only naturally
inactive human calpain (FIG. 1), represents a unique factor
regulating skeletal muscle mass240. Capn6 gene-disrupted
mice exhibit skeletal muscle hypergenesis, and micewith myostatin (Mstn) gene disruption exhibit increased
muscle mass240,241. Therefore, counteracting CAPN6
and/or its downstream molecules, which largely remain
elusive, by specific antibodies or siRNA may ameliorate
the phenotypes of muscular dystrophies. The effect of
CAPN6 inhibition on muscular dystrophies, which mayvary depending on the genes responsible for disease, themodel animal and/or the methodology of inhibition, as
has been reported for myostatin242,243, warrants further
investigation.

Challenges in calpain-targeted therapies
The conventional calpains are expressed in almost allcells. However, because their physiological roles are
generally auxiliary, their inhibition does not cause serious problems under normal conditions. This finding
explains the rationale for using conventional calpain
inhibitors to treat various diseases that are aggravatedby calpain activity. However, calpain activity is required
for some processes that involve the orchestration ofmultiple molecular functions. Therefore, the safety ofchronically inhibiting calpain activity must be considered. Indeed, detrimental effects of calpain inhibition
on cardiomyocytes59,121, the immune system120,123,244,245,
uterine implantation246 and cancer suppression150,155–159
have already been reported (TABLE 3). The use of calpain
inhibitors and potential side effects must also be studied under various conditions: for example, under a perturbed immune system due to disease, the environment
and/or ageing.
Another issue is that in some studies of acute disorders, such as cardiovascular disorders, the calpain inhibitors are administered ex ante, which does not happen in
actual therapy. The effects of calpain inhibition initiatedafter disease onset would be of more practical value.
Except for preventing the effects of infectious diseases or
the manifestation of disease symptoms, most therapies
target calpains at the postsymptomatic stage. To identify
useful clinical treatments, the efficacy and safety of these
therapies should be evaluated using model systems thatreflect the symptomatic context of the disease.
As discussed above, achieving specificity when targeting calpains is challenging. Indeed, many of the first-generation calpain inhibitors were nonspecific and targetedother proteases118. A calpain inhibitor can be useful for
disease treatment, for purely scientific experiments orboth. The difference is exemplified by in vitro specificity versus practical in vivo effects198. In vitro specificity
is gradually being addressed by inhibitor chemistry
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inspired by structures and other new approaches (seeabove and BOX 1). Promising strategies include restricting secondary structures of inhibitors138,139,188,192, learningfrom the 3D structure of calpain–CAST complexes188,192,
allosteric inhibition, including the idea of intercalation
between PC1–PC2 or PEF(L)–PEF(S), and use of cathepsin-specific inhibitors247,248.
Understanding calpain substrate specificities isanother key concern, particularly when considering
potential off-target effects when attempting to target calpains. However, this knowledge is lacking, and solving
calpain substrate specificity remains a key priority in thefield. As discussed above, CysPc domains are structurally
nonspecific with respect to recognizing substrate aminoacid residue side chains64,65. Although bioinformatics
studies have achieved practically usable predictors forcalpain substrate cleavage sites98–102, a constitutive principle of how calpains recognize substrates is far from clear.
Another consideration is that in vivo, calpain inhibition alone may not be completely effective in treating a
disease. Rather, a strategy combining calpain inhibitionand other therapeutic protocols may be more beneficial.
For this approach, the molecular context of functions of
the targeted calpain, including the presence of other drugs
and the disease condition, needs to be thoroughly examined. The same principle holds true in designing genetherapies, and information about how the responsible
calpains function needs to be constantly updated. Recent
reports showing additive effects of calpain inhibition andother drugs are promising examples of this strategy: forexample, MDL28170 in combination with the existing
ALS therapeutic drug riluzole249, or genetic disruption
plus an HSP90 inhibitor 29.

Conclusions and perspectives
Proteases have proved to be effective targets in various
diseases250. However, the paucity of information about thesubstrate specificity of calpains as a protease family has
severely limited our understanding of calpain functions.
This issue has been appreciably overcome in the past decade or so64,65,75,95,96,98.
When targeting calpains, inhibition, activation andrestoration represent therapeutic options depending on
the disease. Promising strategies and inhibitors found todate could be improved through continued basic research.
Importantly, the specific and selective manipulation of
calpain activities is an increasing research trend. As calpain research proceeds, we need to continuously examine
whether newly developed calpain inhibitors are applicablefor therapy, and whether gene therapies for calpainopathies are a feasible therapeutic option.
The pathological mechanisms of known calpainopathies have also been extensively studied using state‑of‑theart techniques combined with mouse genetics andgenome-wide analyses. An exception is the poorly understood role of CAPN10 in the pathology of type 2 diabetes mellitus, an important research topic with substantial
implications for modern society 251,252.
The targeting of calpains in causative organisms for
infectious diseases is highly challenging because thestructures of these calpain species are markedly divergent
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REVIEWS
from those of conventional calpains. There is therefore an
urgent need to systematically advance this field to aid thedevelopment of potential novel therapies.
Translational research involving calpains is still at thedevelopment stage. To advance, we need to learn more
about the calpains themselves, as well as their impact onvarious physiological systems and molecular pathways
and events. The ambiguous impression we have of calpains may simply reflect the fact that they have not yet
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Acknowledgements

The authors thank all of the laboratory members for their inval‑
uable support, Y. Ogata for thoroughly surveying the clinical
trial studies of calpain-related agents, S. Ishiura for valuableadvice on early clinical studies of calpain inhibitors, and L.
Miglietta and G. Gray for their excellent English editing. This
work was supported in part by Grants-in-Aid for Scientific
Research (KAKENHI) from the Japan Society for the Promotionof Science (JSPS) (JP25440059 to Y.O. and JP15H02389 toH.S.), Open Partnership Joint Projects of the JSPS Bilateral
Joint Research Projects (to H.S.), a Takeda Science Foundation
research grant (to Y.O. and H.S.), the Council for Science,
Technology and Innovation’s Strategic Innovation Promotion
Programme “Technologies for creating next-generation agricul‑
ture, forestry, and fisheries” (funded by BTRAI-NARO to H.S.),and a research grant of The Naito Foundation (to H.S.).

Competing interests statement

DATABASES

The authors declare no competing interests.
MEROPS: http://merops.sanger.ac.uk
RCSB Protein Data Bank: http://www.rcsb.org/pdb

FURTHER INFORMATION
Joint Genome Institute: MycoCosm: http://genome.jgi.doe.
gov/programs/fungi/index.jsf
Leiden Muscular Dystrophy: Calpain‑3 (CAPN3): www.dmd.
nl/capn3_home.html

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O N L I N E O N LY
Author biographies
Yasuko Ono is Associate Director of the Calpain Project, Tokyo
Metropolitan Institute of Medical Science (Igakuken), Japan. Ono
received a Ph.D. in protein science from the University of Tokyo, Japan,
studying CAPN3 in the laboratory of Koichi Suzuki. In 1999, Onobegan postdoctoral training in the field of muscle proteins in the laboratory of Carol C. Gregorio at the University of Arizona, USA, and,in 2004, joined the current research project led by Hiroyuki Sorimachi.
Her major research interests include the role of calpain in muscle with
an emphasis on physiology and evolution.
Takaomi C. Saido is Senior Team Leader, Laboratory for Proteolytic
Neuroscience, RIKEN Brain Science Institute, Japan. Saido received
a Ph.D. from the Graduate School of Pharmaceutical Science, theUniversity of Tokyo, Japan, in 1988 and then became a research scientist at the Tokyo Metropolitan Institute of Medical Science, Japan,
where he mainly worked on the biochemistry and pathophysiology of
calpain. Since moving to RIKEN in 1997, he has been working bothon calpain and Alzheimer disease. The Cast knockout, Capn2 (conditional) knockout and App knock‑in mice generated in his laboratory
are widely used in the research community.
Hiroyuki Sorimachi started working on calpains in 1988 in Koichi
Suzuki’s laboratory at the Tokyo Metropolitan Institute of Medical
Science (Rinshoken/Igakuken), Japan. In 1992, he received a Ph.D.
from the University of Tokyo, Japan, after which he was appointed
an assistant professor, and, in 1997, became an associate professor.
In 2004, he returned to Rinshoken/Igakuken as Project Leader of the“Calpain Project”, and, in 2008, he became a department head. He wasawarded a Lifetime Achievement Award from the FASEB SRC Calpain
meeting in 2016. His research interests include the biochemistry andgenetics of all types of calpains.
Key points• The calpains are a family of proteases with biologically vital functions. However, the mechanistic features of calpains are largely
unknown.
• Calpains have been identified as potential therapeutic targets forvarious types of diseases, including neurodegenerative and cardiovascular disorders, ophthalmic diseases and cancer.
• Many disease phenotypes are ameliorated by calpain inhibition, andsome calpain inhibitors have entered clinical trials.
• Many calpain orthologues in parasites or microorganisms are
responsible for the pathogenicity and viability of the organism;
thus, targeting these calpains is a promising approach for combatting infectious diseases.
• Some calpain gene defects resulting in loss of calpain activity arepathologically implicated in human disease. Therefore, in addition
to therapies that inhibit calpain activity, developing strategies thatcompensate for calpain loss are an important goal.
• The development of inhibitors with improved efficiency and specificity for calpains is a critical future research direction. Unveilingthe physiological functions of calpains at the molecular level is a
key challenge.
Subject categories
Biological sciences / Drug discovery[URI /631/154]
Biological sciences / Chemical biology / Proteases[URI /631/92/468]
Health sciences / Diseases / Neurological disorders / Neurodegenerative

diseases[URI /692/699/375/365]
Health sciences / Diseases / Cardiovascular diseases[URI /692/699/75]
Health sciences / Oncology / Cancer[URI /692/4028/67]
Health sciences / Diseases / Infectious diseases[URI /692/699/255]
ToC blurb

research for drug discovery:000 Calpain
challenges and potential
Yasuko Ono, Takaomi C. Saido and Hiroyuki
Sorimachi
The calpain family of proteases are involved innumerous physiological and pathological processes.
Here, Sorimachi and colleagues provide an overview
of the calpain superfamily and calpain-relateddisorders, assess the various emerging approaches fortherapeutically targeting calpains and highlight agentscurrently in clinical trials.

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In format provided by Ono et al. (doi:10.1038/nrd.2016.212)

Supplementary Figure 1. Calpain inhibitors and related molecules
1. leupeptin

2. E-64

3. E-64c

4. E-64d (loxistatin)

5. ALLNal (CI-I, MG101)

6. ALLMal (CI-II)

7. MDL28170 (CI-III)

8. calpeptin

9. MG132

10. CI-IV

11. AK269

12. AK275 (CI-X)

13. CI-V

14. mCalp-I (No.18)

15. AK295 (CI-XI)

16. CI-XII

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In format provided by Ono et al. (doi:10.1038/nrd.2016.212)

Supplementary Figure 1 (continued)
17. PD150606

18. PD151746

19. SJA6017 (CI-VI)

20. CEP-3122(D)

21. MDL104903

22. BDA-410

23. SJA7019

24. SJA7029

25. SNJ1715

26. SNJ1757

27. SNJ1945

28. SNJ2008

29. A-705239(BSF 409425)

30. A-705253 (BSF 419961, CAL 9961)

31. BN 82270

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In format provided by Ono et al. (doi:10.1038/nrd.2016.212)

Supplementary Figure 1 (continued)
32. C-101 (Myodur)

33. C-201 (Neurodur)
Cl-

Cl-

34. GABAdur

35. olesoxime (TRO19622)

36. hypervalent organotellurium compound (No.11, RF19)

37. macrocyclic aldehyde (CAT811)

38. indole-containing 18-membered aldehyde (No. 1c)

39. α-helical peptide (No.3c)

40. dipeptidyl α,β-unsaturated ester (No.8)

41. macrocyclic β-turn peptide (c*[PGALK])NATURE REVIEWS | DRUG DISCOVERY

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Supplementary figure legend
Supplementary Figure 1: Calpain inhibitors and related molecules
Examples of calpain inhibitors and related molecules are numbered here approximately in the orderof their discovery/synthesis (the earliest discovered are at the top, as in Supplementary Table 1).
Note that they are not actually specific for calpains. Descriptive names of the smaller molecules are
as follows (for their properties, see Supplementary Table 1):
1. leupeptin, Ac-L-Leu-L-Leu-L-argininal1
2. E-64, [(2S,3S)-3-carboxyoxirane-2-carbonyl]-L-Leu-(4-guanidinobutyl)amide)2,3
3. E-64c, [(2S,3S)-3-carboxyoxirane-2-carbonyl]-L-Leu-(3-methylbutyl)amide4
4. E-64d (loxistatin), [(2S,3S)-3-ethoxycarbonyloxirane-2-carbonyl]-L-Leu-(3-methylbutyl)amide)4
5. ALLNal (calpain inhibitor (CI-) I, MG101), Ac-L-Leu-L-Leu-L-norleucinal5
6. ALLMal (CI-II), Ac-L-Leu-L-Leu-L-methional5
7. MDL28170 (CI-III), Z-L-Val-L-phenylalaninal6
8. calpeptin, Z-L-Leu-L-norleucinal7
9. MG132, Z-L-Leu-L-Leu-L-leucinal8
10. CI-IV, Z-L-Leu-L-Leu-L-Tyr-CH2F9,10
11. AK269, Z-L-Leu-L-Phe-CONH-C2H511-13
12. AK275 (CI-X), Z-L-Leu-L-Abu-CONH-C2H511-15
13. CI-V, morpholinoureidyl-L-Val-L-homophenylalanyl-CH2F16
14. mCalp-I (No. 18), Z-L-Leu-L-Abu-CONH-CH2-C6H3-3,5-(OCH3)213
15. AK295 (CI-XI), Z-L-Leu-L-Abu-CONH-(CH2)3-morpholine12,13,17
16. CI-XII, Z-L-Leu-L-norvaline-CONH-CH2-2-pyridyl18
17. PD150606, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid19
18. PD151746, 3-(5-fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid19
19. SJA6017 (CI-VI), 4-fluorophenylsulfonyl-L-Val-L-leucinal20
20. CEP-3122, CH3-SO2-D-phenylmethylserine-L-phenylalaninal21(CEP-3453 is the HSO3 addition of CEP-312222)
21. MDL104903, [1-[(5-hydroxy-4-phenyl-methyl-3-oxazolidinyl)carbonyl]-2-ethylpropyl]carbamic
acid phenylmethyl ester23
22. BDA-410, (2S)-N-[(1S)-1-[(S)-hydroxy(3-oxo-2-phenyl-1-cyclopropen-1-yl)methyl]-2-methyl
propyl]-2-benzenesulfonylamino-4-methylpentanamide24-26
23. SJA7019, chloroacetic acid N’-[6,7-dichloro-4-(4-methoxyphenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl]hydrazide27
24. SJA7029, chloroacetic acid N’-(6,7-dichloro-4-phenyl-3-oxo-3,4-dihydroquinoxalin-2-yl)
hydrazide27
25. SNJ1715, (2S)-4-methyl-2-(3-phenylthioureido)-N-[(3S)-tetrahydro-2-hydroxy-3-furanyl]
pentanamide28
26. SNJ1757, (2S,5S)-5-benzyl-6-hydroxy-2-(2-methylpropyl)-3-morpholinone29
1

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27. SNJ1945, [(1S)-1-([((1S)-1-benzyl-3-(cyclopropylamino)-2,3-dioxopropyl)amino]carbonyl)-3methylbutyl]carbamic acid 5-methoxy-3-oxapentyl ester30
28. SNJ2008, [(1S)-1-[([(1S)-1-benzyl-3-(cyclopropylamino)-2,3-dioxopropyl]amino)carbonyl]-3methylbutyl]carbamic acid 2-(pyridin-2-yl)ethyl ester31
29. A-705239 (BSF 409425), N-(1-carbamoyl-1-oxohex-1-yl)-2-[E-2-(4-dimethylaminomethyl
phenyl)ethen-1-yl]benzamide32
30. A-705253 (BSF 419961, CAL 9961), N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethyl
aminomethylphenyl)ethen-1-yl]benzamide32
31. BN 82270 (No. 7), phenothiazine-L-Leu-2-hydroxytetrahydrofuran33
32. C-101 (Myodur), L-aminocarnitylsuccinyl-L-Leu-L-argininal dichloride34 (Counter ions (Cl-) aredepicted as associated, but not covalently connected, objects in the structures).
33. C-201 (Neurodur), L-cysteyl-L-Leu-L-argininal35 (CYLA is diethyl acetal of C-20136)
34. GABAdur, pregabalin-L-Leu-L-argininal37
35. olesoxime (TRO19622), (3Z)-N-hydroxycholest-4-en-3-imine38
37. macrocyclic aldehyde (No. 2d, CAT811), ((7S,10S,13S)-7-formyl-10-isobutyl-9,12-dioxo-2-oxa-8,11-diaza-bicyclo[13.2.2]nonadeca-1(18),15(19),16-trien-13-yl)-carbamic acid benzyl ester39
38. indole-containing 18-membered aldehyde (No. 1c), (S)-N-[(S)-4-methyl-1-oxopentan-2-yl]-2,
16-dioxo-3,20-diazabicyclo[15.2.1]icosa-l(19),17-diene-4-carboxamide40
39. dipeptidyl α,β-unsaturated ester (No. 8), (E)-ethyl-4-(2-(benzyl-oxycarbonyl-amino)-4-methyl
pentanamido)-5-phenylpent-2-enoate41.
For the following larger molecules, molecular ID used in the first paper reporting their synthesis is
as follows:
36. hypervalent organotellurium compound, No. 1142 or RF1943
39. α-helical peptide (Ac-IPPKYCELLC-NH2), No. 3c44
41. macrocyclic β-turn peptide (PGALK), c*[PGALK]45.
Stick representations (C: grey, N: blue, O: rouge, F: lime, S: yellow, Cl: green, I: dark red, Te:turquoise, H: not shown) and 2D chemical structures were energy minimized and drawn by MOE
Ver. 2015.10. In the structure of 39 and 41, a ribbon scheme and/or hydrogen bonds are shown.
Abbreviations: Abu, α-aminobutylic acid residue; Ac, acetyl; Z, benzyloxycarbonyl.

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Supplementary table
Supplementary Table 1: Properties of calpain inhibitors
Ki and IC50 values vary substantially depending on the reference. The values shown here are from the referencesindicated. Numbers (No.) correspond to those in Supplementary Figure 1.

Leupeptin

Composition
FormulaC20H38N6O4

426.56 1969

R

2

E-64

C15H27N5O5

357.41 1978

I

3

E-64c

C15H26N2O5

314.38 1980

I

4

E-64d (loxistatin) C17H30N2O5

342.44 1986

I

5

ALLNal (CI-I,
MG101)

C20H37N3O4

383.53 1986

R

6

ALLMal (CI-II)

C19H35N3O4S

401.57 1986

R

7

C22H26N2O4

382.46 1988

R

8

MDL28170 (CIIII)
Calpeptin

C20H30N2O4

362.47 1988

R

9

MG132

C26H41N3O5

475.63 1991

R

10

CI-IV

C30H40N3O6F

557.66 1991

I

11

AK269

C26H33N3O5

467.57 1993

R

12

AK275 (CI-X)

C21H31N3O5

405.50 1993

R

13

CI-V

C21H30N3O4F

407.49 1994

I

14

mCalp-I (No. 18)

C28H37N3O7

527.62 1996

R

15

AK295 (CI-XI)

C26H40N4O6

504.63 1996

R

16

CI-XII

C26H34N4O5

482.58 1996

R

17

PD150606

C9H7O2SI

306.12 1996

R

18

PD151746

C11H8NO2SF

237.25 1996

R

19

SJA6017 (CI-VI)

372.46 1997

R

20

CEP-3122

C17H25N2O4S
F
C20H24N2O5S

404.49 1998

R

21

MDL104903

C23H28N2O5

412.49 1999

R

22

BDA-410

C26H32N2O5S

484.62 1999

R

23

SJA7019427.67 1999

I

24

SJA7029397.65 1999

I

25

SNJ1715

C17H13N4O3
Cl3
C16H11N4O2
Cl3
C17H25N3O3S

351.47 2003

R

26

SNJ1757

C15H21NO3

263.34 2003

R

No.

Inhibitor

1

Mr

Year*1 Mode*2

Inhibitory activity (upper: Ki, lower: IC50 if otherwise indicated) (nM)
Ref.
C1*3
C2*3
papainCathB*4 CathL
Trypsin Others
3
1,5,20,
320
430
6
35
plasmin: Ki=3.4x10
29,46,47351
176
1.5x103 27
kalikrein: Ki>1.9x105
2,48
800*5
1.0x1032.6x103 290
800
3,4,48960*5
1.5x104 *5
4.4x103 24090
4(active only afterhydrolysis (=E-64c))
86
192
2.2x103 22>5x105 thermolysin: Ki >5x105 5,19,49
310
160
4.5x103 MMP2: IC50=2.19x104
calcineurin/CaM: Ki>2x1055250
140
3.6x1036,46,50725>2x106 plasmin: Ki=2.7x105
100
kalikrein: Ki>5x106
Proteasome: Ki=1.1x105 7,51
52
34
138
Immunoproteasome:
Ki=1.0x103
8,51,52
10
20S proteasome:
1.2x103
IC50=100, Ki=6.9x103
9,10
k*6=
k=
2.9x104 *5
6.8x105
11,18
200
39
4.5x104 6.0x103
11,18
250
210
9.3x104 2.4x103
16,53
ca.70% inhibition of~10~100
total cysteine proteaseactivity from rat kidneyby oral administration(10.0mg/kg).
18,54
940
25
2.3x10322
1.8x1031814041
6.9x103
18
19
120
750
210
370
>5x1051.3x105>5x105 thermolysin: Ki=2.0x105 19,49
MMP2: IC50=9.3x103
calcineurin/CaM: Ki=1.3x104
260
5.3x103 >5x105>2x105>5x105 thermolysin:Ki >5x105 19
calcineurin/CaM: Ki>8.5x104
20,28,29
20S proteasome:
22
49
6.9
IC50>105
21,22
8
5
32
IC50 of CEP3453 for
C1: Ki=15, for CathB:
Ki=15
23
33
24,25,55130
630
thrombin, CathG,
21
21
400
1.6x105proteasome: Ki=1.0x105
CathD: Ki=9.1x105
2777
64
1.5x10327
120
170
4.2x10328
20S proteasome:86
190
IC50>10529
700
930>1x105

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SNJ1945

C25H37N3O7

491.58 2005

R

28

SNJ2008

C27H34N4O5

494.59 2006

R

29

C25H31N3O3

421.54 2003

R

C30H33N3O3

483.61 2003

R

31

A-705239 (BSF
409425)
A-705253 (BSF
419961, CAL
9961)
BN 82270

C25H29N3O5S

483.59 2004

R

32

C-101 (Myodur)

C23H47N7O6Cl2C-201 (Neurodur) C15H30N6O6S
CYLA
C19H40N6O7S

588.58 2005

R

422.51 2005
496.63 2006

R
R

34
35

GABAdur
Olesoxime(TRO19622)

C20H38N6O5
C27H45NO

442.56 2007
399.66 2007

R
R

36

Hypervalentorganotellurium
compound (No.
11, RF19)
Macrocyclic
aldehyde(CAT811)
Indole-containing
18-memberedaldehyde (No.1c)α-helical peptide(No. 3c, AcIPPKYCELLCNH2)
Dipeptidyl α,βunsaturated ester(No.8)

C15H13OCl3Te 443.23 2009

I

200*8

C29H37N3O6

523.63 2009

R

220

30

C25H39N3O4

445.60 2012

R

42

66

C64H96N12O14 1321.67 2012
S2

R

1x104
-

C27H34N2O5

466.58 2012

R

Macrocyclic βturn peptide(c*[PGALK])C30H54N8O6

622.81 2016

R

30

33
-

37

38

39

40

41

170
29
13
27
-

In format provided by Ono et al. (doi:10.1038/nrd.2016.212)

30

99
17

31,56

27
62
-

22
149
-

>1x103

*7

*7

k=
k=
7.9 x103 9.4x103>5x105>1x105-proteasome: Ki=2.6x104 32,57
33,58
Dual inhibitor for
calpains (active afterhydrolysis of the acetylgroup) and lipid
peroxidation. Cellularcalpain inhibition:
IC50=1.33x104; Fe2+
induced lipid
peroxidation in rat brain
microsomes inhibition:
IC50=1.55x104;
permanent hearing andhair cell loss of guinea
pigs induced by soundtrauma: IC50=4x103
spectrin breakdown (145 34
kDa): IC50=3.7x104
35(no Ki or IC50 data)
Total calpain amounts in 36
spinal cord homogenates
were reduced ca. a half
after 2 mg/mouse/day
administration. (active
only after hydrolysis(=C-201))
37(no Ki or IC50 data)
38,59,60
mitochondrialtranslocator protein 18
kDa and its ligand,
PK11195, binding:
IC50=3~5x104
42,43
CathS: k=2.0 x105
CathK: k=3.8 x105

pepsin and αchymotrypsin:
IC50>5x105
α-chymotrypsin:
IC50>2.5x105

70>1x105-proteasome: Ki=4.0x105 32,57

3.9x104-

1.7x104-(~10%)

40

44

IC50 for P. falciparum
growth rate in humanerythrocytes=5x103
IC50 for HeLa cell
growth=3.5x105(~50%*9)

39

41

45

(~100%)*1: a year of the first report for synthesis or discovery; *2: a mode of inhibition, reversible (R) or
irreversible (I); *3: C1, calpain-1; C2, calpain-2; *4: Cath, cathepsin; *5: for chicken calpain-11; *6:
a rate constant (M-1s-1) of a reaction, E+IEI (E: enzyme, I: inhibitor); *7: showing in vivo
inhibitory activity; *8: for P. falciparum proteases; *9: relative to inhibitory effect observed for C2
under the same condition.

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Notes for references
1

In the original report, leupeptin was shown to have DL-argininal; however, a later study61 showedthat L-argininal, but not D-argininal, has a strong affinity to trypsin. Thus, the structure is shown as
Ac-L-Leu-L-Leu-L-argininal.
5

The original report was by Saito, M., Higuchi, N., Kawaguchi, N., Tanaka, T. and Murachi, T. at the
4th FAOB Congress in Singapore, November 30, 1986 (Abstracts of Papers, p58).

11,18

The AK275 was first synthesized as a diastereometric mixture (called CX275); however, since alater study14 showed that the L, L isomer has inhibitory activity, this active structure is shown here,
and so are for other AK series inhibitors.
16

This reference described synthesis and the effect of morpholinoureidyl-L-Leu-L-homophenylalanylCH2F (mu-L-hF-fmk, P34089), but not those of Val (mu-V-hF-fmk, i.e., CI-V). Since the describedmethod can be applied to mu-V-hF-fmk (CI-V) and no other reference describing the synthesis of CIV was found, this reference is cited here.
27

The original report was by Inoue, J., Cui, Y.-S., Sakai, O., Nakamura, M., Yuen, P.-W., and Wang,
K.K.W. (1999) α-Substituted hydrazides having calpain inhibitory activity, in Proceedings of the
FASEB Summer Conference on Calpains, 1999 June 20–23, Breckenridge, CO.
34

This reference did not mention about the stereochemical analysis, and, thus, the structure is an
estimate from those of L-carnitine and leupeptin.

35This reference did not mention about the stereochemical analysis, and, thus, the structure is an
estimate from those of L-cysteic acid and leupeptin.

37

This reference did not mention about the stereochemical analysis, and, thus, the structure is an
estimate from those of pregabalin and leupeptin.

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