Current Medicinal Chemistry, 2000, 7, 1171-1188 1171

A Survey of Calpain Inhibitors

I. O. Donkor*

Department of Pharmaceutical Sciences, The University of Tennessee Health Sciences Center, Memphis,
TN 38163, USA

Abstract: Calpain is unique among the cysteine protease family of enzymes in that it combines thiol
protease activity with calmodulin-like activity. Despite its wide spread distribution the exact physiological
function(s) of calpain is yet to be deciphered. The enzyme is however, implicated in a number of
pathophysiological conditions. Due to the potential of calpain as a therapeutic target a number of inhibitors
have been described for the enzyme. In this article we have grouped calpain inhibitors into those derived from
natural sources, and those derived from chemical synthesis. Additionally, an overview of functional groups
that have been used as “warheadsö of calpain inhibitors is presented along with a discussion of the structure
activity relationship studies of the address region of peptidyl calpain inhibitors. Recent work in this area has led
to a better understanding of the structural requirements for tight binding of inhibitors to the active site of
calpain. A discussion of peptidomimetic calpain inhibitors, nonpeptide calpain inhibitors, and selectivity
of some calpain inhibitors are also presented. The recent disclosure of the crystal structure of a nonpeptide
calpain inhibitor bound to a hydrophobic pocket on the calcium-binding domain of calpain has opened the
door to future development of potent cell permeable nonpeptide calpain inhibitors.

I. INTRODUCTION et al. [4] which disclosed the crystal structure of the

unactivated from calpain II. The crystal structure
Calpain is a member of the cysteine protease revealed that the catalytic triad is not assembled.
family of enzymes. It is unique among this class of Thus, Ca2+-binding must induce conformational
proteins in that it requires Ca2+ for activation. At
least six isoforms of calpain have been identified changes that lead to assembling of the activated
[1]. Two of the major calpain isoforms known as enzyme.
calpain I (µ-calpain) and calpain II (m-calpain) are
widely distributed in mammalian cells. These Calpain has a broad range of substrate specificity
isoforms are very identical and differ mostly in and a number of cellular proteins are calpain
their Ca2+ requirement for activation. Calpain I is substrates. These include calmodulin-binding
activated by micro molar concentration of Ca2+ (1- proteins, cytoskeletal proteins (e.g., spectrin,
100 µM) whereas calpain II requires millimolar MAP-2), G-proteins, enzymes involved in signal
concentration of Ca2+ (0.1-1 mM) for activation in transduction (e.g., PKC, IP3 kinase), membrane
vitro. The presence of phospholipids make calpain receptors (e.g., EGF receptor), and transcription
more sensitive to Ca2+ thus lowering the factors [5-10]. Most calpain substrates possess
concentration of Ca2+ required for activation in vivo hydrophilic residues such as proline, glutamic
[2]. Structurally, calpain is a heterodimeric protein acid, aspartic acid, serine, and threonine (i.e. the
consisting of a cysteine-proteinase domain coupled PEST sequence) close to the cleavage site.
to a calmodulin-like Ca2+-binding domain. Calpains
I and II consist of an 80 kDa and a 78 kDa catalytic Calpain plays a role in several intracellular
subunit (large subunit) respectively, with each signaling pathways mediated by Ca2+ but the
subunit coupled to a 30 kDa regulatory subunit precise function(s) of the enzyme in vivo awaits
(small subunit). The crystal structure of the clarification. Calpain is implicated in a number of
regulatory subunit was the first to be solved [3]. pathological conditions and the enzyme has been
This was recently followed by the work of Hosfield proposed as a potential therapeutic target for a
number of diseases including brain trauma, spinal
*Address correspondence to this author at the University of Tennessee, cord injury, Alzheimer’s disease, Parkinson’s
Memphis, 847 Monroe Avenue, Faculty Bldg. Room 327E, Memphis, TN disease, subarachnoid hemorrhage, muscular
38163, USA; Phone: (901) 448-7736; Fax: (901) 448-6828; Email: dystrophy, cardiac ischemia, restinosis, thrombotic
[email protected]
platelet aggregation, arthritis, and cataract. Several
review articles and books provide detailed

0929-8673/00 $19.00+.00 ¬ 2000 Bentham Science Publishers Ltd.

1172 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

description of calpain structure, physiological pyrazinone derivatives are among the calpain
functions, and potential as a therapeutic target [6, inhibitors isolated from Streptomyces species.
11-21]. In this Review Article calpain inhibitors Leupeptin (Ac-Leu-Leu-Arg-H) [30], antipain
derived from natural and synthetic sources are ([(S)-1-carboxy-2-phenylethyl]-carbamoyl-L-Val-
discussed with emphasis on the structure-activity Arg-H) [31], strepin P-1 (N-i-valeryl-Tyr-Val-Arg-
relationship (SAR) of the inhibitors. H) [32] and the pentapeptides staccopins P-1 and
P-2 (Val-Val-Val-Val-Phe-H and Val-Val-Val-
Val-Tyr-H, respectively) [33] are examples of
II. CALPAIN INHIBITORS peptidyl aldehyde calpain inhibitors isolated from
Streptomyces. The inhibitors inactivate calpain by
The search for calpain inhibitors has been an on reacting reversibly with the active-site thiol of the
going process following the discovery of the enzyme but they are not selective for calpain. For
enzyme over 30 years ago [22, 23]. General example, leupeptin inhibits plasmin, trypsin,
protease inhibitors such as EDTA and EGTA (Ca2+ papain, and cathepsin B; strepin P-1 inhibits
chelators), iodoacetic acid, 5,5’-dithiobis(2- papain and trypsin; staccopins P-1 and P-2 are
nitrobenzoic acid), N-ethylmaleimide, mersalyl, however, not active against serine proteases but
isocoumarins, and diisopropyl phosphorofluoridate they are potent inhibitors of calpain and papain.
are among the first inhibitors of the enzyme to be The lack of selectivity and poor cellular
identified. Most of these inhibitors are neither permeability make the inhibitors unattractive as
selective nor potent which make them unattractive biomedical tools for investigating the role(s) of
as biomedical tools for studying calpain-mediated calpain in cell function. In attempts to improve the
potency and cell permeability of peptidyl
events. Current knowledge about calpain substrate aldehydes, SAR studies involving N-terminal
specificity and the nature of the active site of the capping with lipophilic substituents were
enzyme have made possible the development of undertaken. The studies led to the discovery of
potent and in some cases selective calpain calpeptin (Z-Leu-Nle-H) which possesses a
inhibitors. benzyloxycarbonyl moiety as the lipophilic N-
terminal agent [34]. The compound is cell
A. Calpain Inhibitors Derived From permeable and inhibits human platelet calpain II with
Natural Sources an IC50 of 40 nM [35]. Incorporation of the
benzyloxycarbonyl group into Val-Phe-H gave
i. Calpain Inhibitors Derived from MDL 28,170 (1), Fig. (1), which is also cell
Mammals permeable [36]. Using an acetyl moiety as the N-
terminal substituent, Sasaki et al. [37] discovered
Calpastatin is an endogenous polypeptide that calpain inhibitor I (Ac-Leu-Leu-Nle-H) and
specifically inhibits both major isoforms of calpain calpain inhibitor II (Ac-Leu-Leu-Met-H). The
but not papain or cathepsin B [24]. The polypeptide inhibitors are however, not specific for calpain.
consists of an N-terminal domain (domain L) and Calpain inhibitor I is a better inhibitor of cathepsin
four repetitive domains (repeats 1-4). All four L (Ki = 0.5 nM) and calpain inhibitor II inhibits
repeats have similar inhibitory activity against cathepsin B (Ki = 100 nM). Sasaki and coworkers
substituted 4-phenylbutylryl on the N-terminal
calpains I and II but domain L is devoid of nitrogen of Val-Phe-H to obtain compound 2
inhibitory action [25]. A synthetic 27-residue which inhibited calpain I and calpain II with Ki of
peptide derivative based on a repeat region of 36 nM and 50 nM, respectively. Compound 2
calpastatin is also an inhibitor of calpain [26]. Other weakly inhibited trypsin, chemotrypsin, and
polypeptide inhibitors of calpain are α2- cathepsin H [37]. Thus, starting with peptidyl
macroglobulin [27] and the heavy chains of L- and aldehydes isolated from Streptomyces, SAR
H-kininogen [28]. Puri et al. [29] employed the studies involving N-terminal capping led to
highly conserved sequence, Gln-Val-Val-Ala-Gly- compounds with enhanced cell permeability and
NH2, present in domains II and III of human calpain inhibitory potency. The compounds,
kininogens as an address region to which they however, still lack specificity and are readily
appended a 3-nitro-2-pyridyl moiety via a disulfide oxidized under physiological conditions because
linkage to generate a weak irreversible inhibitor of they are aldehydes [38].
platelet calpain I. Omura et al. [39] isolated cystamidin A (3) from
Streptomyces species KP-1241 culture. They
ii. Calpain Inhibitors Derived from Plants claimed that the compound is a calpain inhibitor but
Streptomyces species is a rich source of calpain provided no calpain inhibitory data. Bihovsky and
inhibitors. Peptidyl aldehyde, diketopiperazine, and Pendrak [40] subsequently synthesized cystamidin
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1173

O O
H H
O N H N H
N N
H H
O O O O

M D L 28 ,1 70 ( 1) 2
HO
OH
O O
HN
O
NH 2 N
N
N
H N
O
3 4
5
O O

HO C HO

OH
O O O O O
H 7
N N O
N N OH
H H
O O
O
HO
6 HO
8
OH

Fig. (1). Calpain inhibitors derived from natural sources.

A (pyrrole-3-propanamide) and its regioiosmer inhibited calpain with an IC50 of 7.1 µM but it did
(pyrrole-2-propanamide) and found the compounds not inhibit papain at 200 µM.
to be inactive against recombinant human calpain I.
Alvarez et al. [41] isolated 3-benzyl-6-isopropyl Peptidyl epoxysuccinate inhibitors are the most
disubstituted 2(1)-prazinone 4 from Streptomyces studied group of cysteine protease inhibitors
sp. The compound (which was termed Phevalin) is isolated from microbes. Trans-epoxysuccinyl-L-
presumably derived biosynthetically from leucylamido-4-guanidino-butane (E-64) (9, Table
phenylalanine and valine. Phevalin inhibited calpain 1) was the first member to be reported. It was
activity in the casein hydrolysis assay with an IC50 isolated from Aspergillus japonicus TPR-64 by
of 1.3 µM. Diketopiperazine 5 (IC50 = 0.8 µM) Hanada et al. [45] and immediately followed by its
and tetrapeptide 6 (IC50 = 1.2 µM) are new calpain total synthesis and structure characterization [46].
inhibitors bearing N-methyl tyrosine residues that E-64 and its derivatives inactivate cysteine
were isolated from Streptomyces griseus (SC 488) proteases by forming a covalent bond with the
[42]. Damnacanthal (7), an anthraquinone active site thiolate. Epoxysuccinyl peptides are
compound isolated from the root of Morinda highly selective for cysteine proteases and they
citrifolia effectively inhibited thrombin and calpain. have been used as active site titrants for this class of
The compound is not selective since it inhibited enzymes [45-50]. The compounds display no
both serine and cysteine proteases [43]. Chung et selectivity for calpain relative to the other cysteine
al. [44] isolated penicillide 8 having a 5H,7H- proteases [47]. The mechanism for irreversible
dibenzo[b,g][1,5]dioxocin-5-one skeleton from the inactivation of cysteine proteases by
culture broth of Penicillium species F60760 as a epoxysuccinates and their mode of binding to
nonpeptide inhibitor of calpain. The compound cysteine proteases have been studied by NMR [51]
1174 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

Table 1. Epoxysuccinyl Derivatives have found that such cyclopropyl derivatives of E-

64c are devoid of calpain inhibitory activity
O
attesting to the importance of the epoxide ring for
the bioactivity of this class of compounds [56]. b).
R1O
R2 The stereochemistry of the epoxide group is
X important since the L-isomer of E-64 was a better
O
inactivator than the D-isomer [53]. c). Replacement
of the charged guanidino group at P3 with an
Compd. R1 X R2
isopentyl moiety as in Ep-475 did not affect
inhibitory potency but improved cellular
E-64 (9) H O Leu- permeability [53, 57]. d). N-Terminal capping with
NH(CH2)4NHC(NH)NH2 a benzyloxycarbonyl group, as in Ep-460 (15,
Ep-475 H O Leu-NH(CH2)2CH(CH3)2
Table 1), resulted in a dramatic increase in the rate
(10) of inactivation suggesting that calpain has a
hydrophobic binding site that can accommodate this
DC-11 (11, see structure below)
group. The significance of the carboxylate group
12 Et NH Leu-OBzl attached to the epoxide ring has been investigated.
13 Et N-Phe-Boc Leu-OBzl
Esterification of this group (as in compound 16)
resulted in 100-1000 fold decrease in calpain
14 H CH2 Leu-NH(CH2)2CH(CH3)2 inhibition. The esterified compounds however,
Ep-460 H O Leu-NH(CH2)4NH-Z readily permeated cells and were easily hydrolyzed
(15) to the active acid derivative [58]. Murata et al. [59]
16 Et O Leu-NH(CH2)2CH(CH3)2
disclosed E-64 derivative 17 as a poor inhibitor of
calpain II (IC50 = 200,000 nM) but an excellent
17 Et O Ile-Pro-OH inhibitor of cathepsin B (IC50 = 2.28 nM).
18 H O Phe-NH(CH2)2CH(CH3)2 Giordano et al. [60] synthesized epoxysuccinyl
derivatives with phenylalanine (18),
19 H O Tyr(I)-NH(CH2)2CH(CH3)2
monoiodotyrosine (19), and diiodotyrosine (20) as
20 H O Tyr(I2)-NH(CH2)2CH(CH3)2 the P2 substituent but the compounds were better
inhibitors of cathepsin B than calpain. Replacement
HO OC H O of the epoxide ring with a cyclic sulfate gave 1,3,2-
H
N dioxathiolane dioxide derivatives (21-23, Table 2)
H N which were better inhibitors of cathepsin B than
H
O
calpain [61, 62]. Indeed, cyclic sulfate 21 was
about 430-fold more potent against cathepsin B
D C - 1 1 ( 11 ) than calpain suggesting that the catalytic site of
calpain is sterically more hindered than that of
cathepsin B.
and X-ray crystallography [52]. SAR studies of E- Table 2. Cyclic Sulfate Epoxysuccinyl Derivatives
64 derivatives led to the following observations
(Table 1): a). The epoxide ring is important for
activity. E-64c [Ep-475, (10)] was found to be O
over a thousand fold more effective than DC-11 R OO C H
(11) in which the epoxide ring was replaced by N
N
a H
double bond [53]. Aziridine analogues of E-64c O O O
S
(e.g., 12) are generally poor calpain inhibitors. O O
Substitution on the aziridine nitrogen gave
compound 13, the R,R diastereomer of which was
2-fold and 7-fold selective for calpain I compared to µM)
IC50 (µ
calpain II and cathepsin H, respectively [54]. Compound R Calpain Cathepsin B
Kumar et al. [55] recently reported the
stereoselective synthesis of E-64 derivatives in 21 H 0.3 0.0007
which the cyclopropyl ring was used as a
bioisosteric replacement for the epoxide ring 22 Et 15.0 0.025
(compound 14) but gave no biological data. We 23 Bzl 6.0 0.009
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1175

B. Calpain Inhibitors Derived from Table 3. Reversible Calpain Inhibitor Warheads

Chemical Synthesis
Peptidyl calpain inhibitors consist of an address Warhead E.g. (Calpain inhibition)a Ref.b
region for enzyme recognition and a “warhead” for
covalent modification of the active site thiol. The H
inhibitors are generally designed by replacing the Z-Val-Phe-H (MDL 28170 [36]
scissile amide bond of calpain substrates with an O (IC50 = 0.01 µM)
electron deficient center (the warhead) that is
O
capable of reacting either reversibly or irreversibly
with the active site thiol. In reversible inhibitors the Z-Leu-Phe-COOH [75]
electron deficient center is usually an electrophilic OH
(Ki = 0.0085 µM)
carbonyl containing functional group (such as an O
aldehyde or a ketone). The inhibitors form
reversible covalent abducts (hemithioacetal or O
hemithioketal) with the active site thiolate of the R
Z-Leu-Phe-COOEt [75]
enzyme. The abducts resemble the transition states O
(Ki = 1.8 µM)
or reaction intermediates in substrate hydrolysis so O
they are tightly bound to the enzyme, hence they are
usually effective competitive inhibitors. O

i. Calpain Inhibitor Warheads N HR Z-Leu-Phe-CONH(CH2)2Ph [75]

(Ki = 0.0052 µM)
O
Electrophilic carbonyl containing functional
groups that have been used as warheads in O
OR
reversible calpain inhibitors include aldehydes P
[34-37, 63-74] and α-keto carbonyl compounds OR Z-Leu-Leu-P(O)(OCH3)2 [85]
such as α-ketoacids, α-ketoamides, α-ketoesters (IC50 = 0.43 µM)
O
[75-84], α-diketones [76], and α-keto phosphorus
compounds [85] (Table 3 and Fig. 2). Li et al. [75] O
studied a series of α-keto carbonyl containing P
OR

calpain inhibitors and observed that calpain R Z-Leu-Leu-P(O)(Ph)OEt [85]

inhibitory potency follows the order: α-ketoacids > (IC50 = 0.37 µM)
O
α-ketoamides > α-ketoesters. Using N,N-
disubstituted α-ketoamides they demonstrated that O
the difference in activity between α-ketoamides and P
R

α-ketoesters is due to the inability of α-ketoesters R Z-Leu-Leu-P(O)(CH3)2 [86]

to form a critical H-bond interaction with the (IC50 > 10 µM)
O
enzyme. α-Keto heterocycles such as 24 (IC50 =
90 nM vs. calpain II) and 25 (IC50 = 33 nM) [86-
90] were designed as prodrugs to circumvent the 24 (IC50 = 0.09 µM vs. [86]
inherent metabolic instability of aldehydes in vivo m-calpain)
and also to increase localization of the inhibitor in HO O
neuronal cells. The amphiphilic properties of the
cyclopropenone ring led Ando et al. [91] to design O
cysteine protease inhibitors incorporating this ring
26 (IC50 = 0.35 µM) [91]
as a warhead (e.g. 26, IC50 = 350 nM).
Replacement of the scissile bond of calpain
substrates with epoxysuccinate and its derivatives a
The enzyme is calpain I except where indicated otherwise.
(e.g., aziridine and dioxathiolane) [45-50, 61, 92], b
Selected references.
ketomethylenes [38, 93-100], methylsulfonium
salts [38, 101, 102], vinyl sulfones [103], and calpain relative to serine proteases. The basis for
disulfide linkages [29] afford irreversible inhibitors selectivity may be attributed to the greater
of the enzyme (Table 4 and Fig. 2). Peptidyl nucleophilicity of cysteine (thiolate) versus serine.
epoxysuccinates, acyloxymethyl ketones, and Peptidyl chloromethyl ketones on the other hand
diazomethyl ketones are selective inhibitors of
1176 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

O
H
( H 2 C ) 1 4H 3C N O
S N
O O H
OH O
OH
N
O
O N
H
24 O

M D L 1 04 ,90 3 ( 25 )

O
O
H
N
S N
O O H
OH

26
O
O
H O
O N P
N O
O
H
O O

27

O O

HN +
O N N S
H H
O O

28
O N N
H
O N N
N O
H
O O

29

O O O
O O
H H H
N N O H 3C N N S
N S N
H O H
O O O

O S O

30 31

Fig. (2). Examples of reversible and irreversible calpain inhibitors.

Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1177

Table 4. Irreversible Calpain Inhibitor Warheads

Table 4. Irreversible Calpain Inhibitor Warheads

Warhead E.g. (Calpain inhibition)aa Ref.

Warhead E.g. (Calpain inhibition) Ref.
H OO C
H OO C
Ep-475 (7,450) [53]
Ep-475 (7,450) [53]
O
O
HO OC
HO OC
12 (inactive) [54]
N 12 (inactive) [54]
N
H
H
H OO C
H OO C

O O 21 (IC50 = 0.3 µM) [61,62]

O O 21 (IC50 = 0.3 µM) [61,62]
S
S
O O
O O

N2
N2 Z-Leu-Leu-Tyr-CH=N2 (230,000) [97]
O Z-Leu-Leu-Tyr-CH=N2 (230,000) [97]
O
F
F Z-Leu-Leu-Phe-CH2F (290,000) [94]
O Z-Leu-Leu-Phe-CH2F (290,000) [94]
O
Cl
Cl Z-Leu-Leu-Phe-CH2Cl (9,500) [107]
O Z-Leu-Leu-Phe-CH2Cl (9,500) [107]
O
O
O

O Ar Z-Leu-Phe-CH2OCO-2,6-Cl2-Ph (11,000) [97]

O Ar Z-Leu-Phe-CH2OCO-2,6-Cl2-Ph (11,000) [97]
O
O
O Ar
O O Ar
P O
O P O
O O 27 (365,000) [98,108]
Ar 27 (365,000) [98,108]
O Ar
O
+
+
S
S 28 (200,000) [101]
O 28 (200,000) [101]
O
H et
O H et
O 29 (230,000) [99,100]
O 29 (230,000) [99,100]
O
O O
O S O
S Z-Leu-Leu-Tyr-VSPh (24,300) [103]
Z-Leu-Leu-Tyr-VSPh (24,300) [103]
O2 N
O2 N
O
O
S S Phe-Gln-Val-Val-Cys-Npys (5,850) [29]
S S Phe-Gln-Val-Val-Cys-Npys (5,850) [29]
RH N
RH N
a
Calpain inactivation rate is expressed as M-1s-1 except as indicated for compound 21. VSPh is (vinylsulfonyl)benzene. Npys is 3-nitro-2-sulfenylpyridine.
a proteases calpain inhibition by peptidyl
Calpain inactivation rate is expressed as M -1 s -1 except as indicated for compound 21. VSPh is (vinylsulfonyl)benzene. Npys is 3-nitro-2-sulfenylpyridine.
chloromethyl ketones is due to alkylation of the
inactivate both cysteine and serine proteases. The active site thiolate [104] while inactivation of serine
mechanism of inactivation is however, different for proteases involves alkylation of the active site
the proteases. In concert with other cysteine histidine [105]. In attempts to generate selective
protease inhibitors, peptidyl fluoromethyl ketones
1178 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

were investigated as enzyme inhibitors [106]. studies of the primed region of calpain inhibitors.
Compounds incorporating fluoromethyl ketone The authors also demonstrated that hydrogen
warheads were found to be potent inhibitors of bonding between calpain and the P1-P1’ amide
calpain compared to the corresponding moiety of α-ketoamides is important for enzyme
chloromethyl ketone analogues. For example, Z- inhibition. For example, monosubstituted α-
Leu-Leu-Phe-CH2Cl is a weak inhibitor of calpain ketoamide 33 was 380-fold more effective than
(9,500 M-1s-1) [107] while Z-Leu-Leu-Phe-CH2F N,N-disubstituted α-ketoamide 34. The difference
(290,000 M-1s-1) is a potent calpain inhibitor [94]. in calpain inhibitory potency was attributed to the
Within the diazomethyl ketone class of inhibitors, inability of 34 to form a critical hydrogen bond at
tripeptide derivatives are more potent than dipeptide the active site of the enzyme. In a recent study,
derivatives. For example, Z-Leu-Leu-Tyr-CH=N2 Chatterjee et al. [110] reported a series of potent
is a potent inhibitor of calpain (230,000 M-1s-1) calpain I inhibitors such as 35 and 36 with
while Z-Leu-Tyr-CH=N2 (1,470 M-1s-1) is a weak substituents spanning the S’ subsite of calpain.
calpain inhibitor [97]. Dipeptides with phosphorous
containing ketomethylene warheads (i.e. methyl The S1 subsite of calpain can tolerate a wide
phosphinates, phosphonates, and phosphates) such variety of amino acids at the P1 position of
as 27 (365,000 M-1s-1) [98, 108] and tripeptides inhibitors [75, 94, 111]. For example, P1-Phe (37,
with dimethylsulfonium methyl ketone warheads 136,300 M-1s-1) is preferred over Abu (38, 24,250
such as 28 [101] are potent irreversible calpain M-1s-1), and serine (39, 21,000 M-1s-1) [94].
inhibitors. Compared to these compounds, Protection of the P1-Ser residue as a
acyloxymethyl ketones are weaker inhibitors of tetrahydropyranyl ether as in 40 (100,000 M-1s-1)
calpain while aryloxymethyl ketones are practically increased calpain I inhibition 5-fold. Introduction of
inactive [98, 101, 109]. Sulfonium methyl ketones Tyr(Bn) and Lys(SO2-Ph) at P1 as in compounds
are among the most potent inhibitors of calpain 41 (Ki = 4 nM) and 42 (Ki = 2 nM) resulted in 8-
known [101]. Compared to other warheads, the fold and 12-fold more potent inhibitors than 43 (Ki
order of reactivity towards calpain is: sulfonium = 36 nM), respectively [63]. The results suggest
methyl ketone > fluoromethyl ketone, diazomethyl that the S1 subsite of calpain can accommodate
ketone >> acyloxymethyl ketone [101]. The large hydrophobic groups from the P1 position of
sulfonium methyl ketone warhead is a good motif inhibitors to enhance calpain inhibition. The data
for controlling selectivity through an affinity for the also show that the presence of polar groups such as
S’ subsite of calpain by varying the substituents on the hydroxyl moiety of serine in the S1 pocket is
the sulfur atom. This possibility is however, detrimental to binding. This polar residue
virtually unexplored. Heteroaryloxymethyl ketones intolerance at the S1 subsite might account for the
including pyridyloxy-, pyrimidinyloxy-, 2- dramatic drop in activity observed by Angliker et al.
oxodihydrofuran-4-yl-oxy, benzotriazolyloxy- [112] in their study of Z-Leu-D,L-Tyr-CH2F
methyl ketones (e.g. 29, 230,000 M-1s-1) have (17,000 M-1s-1) and Z-Leu-D,L-Phe-CH2F
been reported as cysteine protease inhibitors [99, (136,300 M-1s-1) and the similar activities of Z-
100]. According to Palmer et al. [103] dipeptide Leu-D,L-Tyr-CH2F and Z-Leu-D,L-Ser-CH2F
vinyl sulfones such as 30 do not inhibit calpain and (21,000 M-1s-1) as inhibitors of chicken gizzard
tripeptide derivatives such as 31 (8,400 M-1s-1) are poor calpain II.
inhibitors of the enzyme.
Until recently, it was a generally held view that
ii. SAR of the Address Region of Calpain to obtain a potent peptidyl calpain inhibitor the P2
Inhibitors residue must be either L-Val or L-Leu. However,
Chatterjee and coworkers [63] used a variety of D-
Various researchers have investigated the amino acids including D-Ser(Bn) (44, Ki = 8 nM),
address region of calpain inhibitors in attempts to D-Thr(Bn) (45, Ki = 9 nM), D-Phe (46, Ki = 11
enhance potency, selectivity and cellular nM), D-Trp (47, Ki = 10 nM), and D-(thiophen-2-
permeability of the inhibitors. Most of the studies yl)-Ala (48, Ki = 11 nM) to demonstrate that
have centered on the unprimed region of the calpain can tolerate D-amino acids at the P2 position
inhibitors. Li et al. [75] used the α-ketoamide of inhibitors. They also showed that 49 with L-
warhead to access both the primed and unprimed Ser(Bn) (Ki = 41 nM) at P2 is more than 5-fold less
subsites of calpain. In this study, ketoamides 32 potent than 44 with D-Ser(Bn) at P2 attesting to the
and 33 Fig. (3) showed 28-fold and 18-fold importance of the D-configuration at P2. Tripathy et
selectivity for calpain II over calpain I, respectively. al. [113] studied the effect of introducing constraint
This finding suggested that calpain isoform in the P2 residue on calpain inhibition using proline
selective agents might be obtained via SAR as the P2 substituent. The enzyme accommodated
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1179

Ph Ph
O O O O
H H
O N O N
N N N N
H H H H
O O O O

33
32
Ph Ph
O O Cl O O
H
O N N H S O 2X
N N N NH
H H
O O O
Cl

34
CN
3 5, X =
S

O R
H
O N
N F 36 , X = N HA c
H S
O O

3 7, R = B n; 3 8, R = Et;
3 9, R = C H 2 O H ; 40 , R = C H 2 O T H P

O R O
(D)
H (D)
R H
O N
H N
H
HN O
NH O
S O2 C H3 C H 3S O 2

4 4, R = C H 2 O B n ; 4 5, R = ( S) ( C H 3 ) C H O B n
4 1, R = C H 2 C 6H 4- p - B n
4 6, R = C H 2 P h; 47 , R = C H 2 - ( 3- i nd ol e )
4 2, R = ( C H 2 )4 N H S O 2P h
4 3, R = C H 2 C H 3 4 8, R = C H 2 - ( 2- th io ph e ne ) ; 49 , R = L - C H 2 O B n

S O2 C H3 S O 2C H 3
N N
H H
N CH O N C HO
H H
O O

50 51

Fig. (3). Examples of compounds used to study calpain active site.

both L-proline (50, IC50 = 45 nM) and D-proline bulky hydrophobic substituents than that of
(51, IC50 = 53 nM) derivatives at the P2 position calpain II as reflected by the apparent second order
further supporting the suggestion that L-Leu and L- rate constant of inactivation of the compounds.
Val are not obligatory substituents at the P2- Further investigation of the bulk tolerance of the
position of a potent calpain inhibitor. Giordano et S2 pockets of the enzymes may lead to the
al. [59] synthesized a series of EP-475 derivatives discovery of calpain isoform selective agents.
incorporating Phe (18), Tyr(I) (19), and Tyr(I2)
(20) in place of the P2-Leu residue of compound
10 (Table 1). This study suggested that the S2
subsite of calpain I is a better accommodator of
1180 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

O O
H H N
O H
O N N N
H N
N Br H
H N
O O H O
O

52 53 54
O

O N O N
O
H
N H
N NH 2
H N N
H O
O H O
N O
H
O

55 56 57

O O

O O
N O O H
H H N
N N H
H H
O O
O O

59
58 62

C H 3S O 2N H
O
H H N
O N N S O
(D ) C H2 C H2 N HS O2 H
+ N
O O S H

O O

60 63

F
O
H H N O
N N S
H
C H 2 C H 2N H S O 2 O N H

F O O
O O

61 64

Fig. (4). Examples of peptidomimetic calpain inhibitors.

SAR studies aimed at generating peptidomimetic bioisosteric replacements of the backbone amide of
replacements for peptide-based enzyme inhibitors dipeptide and tripeptide inhibitors of the enzyme.
have attracted considerable attention due to Most of these changes have focused on the P2 and
pharmacokinetic problems associated with peptidic P3 regions of the inhibitors. The critical role that
inhibitors. The general approach to the design of the P1-P2 amide linkage plays in calpain inhibition
peptidomimetic calpain inhibitors involves [114, 115] generally precludes this region from
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1181

extensive modification. Nonetheless, several and carbamethylene (e.g. 63, IC50 = 30 nM)
oxygen-containing heterocyclic compounds (e.g. inhibitors of recombinant human calpain I
25) with modified P1-P2 amide linkage have been Chatterjee et al. [73] reported that the P2-P3 amide
reported as useful peptidomimetic cysteine protease bond of a potent dipeptide or tripeptide inhibitor of
inhibitors. Such compounds are potent aldehyde calpain is not a strict requirement for calpain
prodrugs with excellent oral bioavailability and inhibition. This finding is at odds with the
cellular permeability [89, 116, 117]. results of Dolle and colleagues [131] who
concluded that the P2-NH moiety is a critical
Azapeptide-based peptidomimetics have been hydrogen-bonding site, which must be maintained in
reported as calpain inhibitors. In these compounds a peptidic calpain inhibitor. Unlike the P2-P3
the alpha carbon of the P1 amino acid residue is amide linkage, the P1-P2 amide bond is very
replaced with a nitrogen atom. Such a modification important for calpain inhibition. Thus,
reduces the electrophilicity of the P1 carbonyl group ketomethylene derivative 64 is 250-fold less potent
and changes the geometry of the alpha position than the parent compound, MDL 28,170 (1)
from tetrahedral to trigonal (i.e. planar, achiral) against chicken gizzard smooth muscle calpain
[118]. This makes azapeptides more resistant to [114]. The loss in potency of 64 reflects the lack of
enzymatic hydrolysis than their normal peptide a critical hydrogen bond interaction between the
analogues. The reduced reactivity of the carbonyl NH of the P1 amide and calpain [115].
group also makes azapeptides with poor leaving
groups poor inhibitors of serine proteases [119, N-terminal capping of the P3 and P4 residues of
120-123]. However, due to the greater calpain inhibitors prevent metabolism by
nucleophilicity of the thiolate at the active site of aminopeptidases, improve cellular permeability,
cysteine proteases, these enzymes including calpain and serve as additional recognition elements for
are inhibited by azapeptides [124]. Thus, binding to the S3 and S4 subsites of calpain. Amino
azapeptide α-halomethyl ketones such as 52 are acid protecting groups such as benzyloxycarbonyl
moderate inhibitors of calpain (1,500 M-1s-1) and [34, 35], acetyl [37], and 4-phenylbutylryl [37]
poor inhibitors of cathepsin B (875 M-1s-1) [125]. have been employed as N-terminal capping agents
of synthetic analogues of calpain inhibitors derived
Peptidomimetic calpain inhibitors incorporating a from microbes as discussed above. Other groups
variety of heterocylces at the P2 position have been that have been used as N-terminal capping agents
reported in the recent literature [see Fig. (4) for [see Fig. (5)] include aryl- (or alkyl) sulfonyl
examples]. Peptide mimetic constructs that have groups (e.g. 65-68, 66, 14, 15) [63, 64, 87-89,
been introduced at the P2 position include 132, 133]; alkanoyl groups (e.g. 69, 70, [65, 66,
substituted quinolinecarboxamides such as 53 132]; various substituted benzoyl, naphthoyl, and
(IC50 = 12 nM), which is equipotent with standard several heterocyclic acyl groups [64, 132]; xanthine
L,L-dipeptide aldehydes [81, 126]; (e.g. 62) [72, 73]; thionaphthalene (e.g. 63) [74,
indolecarboxamides (e.g. 54, IC 50 = 30 nM) 75]; and cyclic sulfonamides like 3,4-
[127]; and ketobenzamides (e.g. 55, Ki < 10 µM) dihydrobenzothiazines (e.g. 71) and related
[84, 128]. 1,4-Disubstituted piperidines (e.g. 56 heterocycles [134].
and 57 and cyclohexanes (e.g. 58) have been used
as scaffolds for the P2-P3 bridge [67, 71, 83].
Aryl-substituted benzoyl groups (e.g. 59) and C. Nonpeptide Calpain Inhibitors
other aryl-substituted benzoheterocycles have been
used as P2 mimetic constructs [82, 129, 130]. Inorganic lead and nonpeptide organic
These examples clearly demonstrate that compounds of diverse structure are calpain
benzoheterocycles are good peptide mimetic inhibitors. Lead (Pb2+) is a viable substitute for
replacements for Leu or Val at P2. Additionally, it Ca2+ in a variety of intracellular processes but it
has recently been demonstrated that replacement of also acts as a noncompetitive inhibitor of calpain
the P2 residue of α-ketoamides such as 60 (Ki = depending on the concentrations of both ions [135].
10 nM) with a 2,6 difluorobenzoyl group as in 61 A series of α-mercaptoacrylates of which 72
(Ki = 14 nM) affords equipotent calpain inhibitors (PD150606) and 73 (PD151746) [Fig. (6)] are
[110]. This study revealed that the binding affinity examples were disclosed by Wang et al. [136,
offered by substituents spanning the P’ address 137] as potent, selective and cell permeable
region of α-ketoamide inhibitors of calpain can uncompetitive (with respect casein) inhibitors of
adequately offset the loss in binding affinity due to calpain. X-ray crystal studies revealed that 72
the absence of a P2-amino acid residue. Using a inhibit calpain by binding to a hydrophobic pocket
series of ketomethylene (e.g. 62, IC50 = 25 nM) on domain VI of calpain small subunit [3].
1182 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

F
O
H O
N H H
S N H3 C N H
O O H S N
O O H
O
O
O
65

66

HO
O O
O
H
N H H
N N
S N
H S N
O O H
O O O

67 68

C H 3( C H 2) 7 CH=CH NH O
O O CH 3
H
H3 C N N H
N N
OH H H
O O
C O NH 2
69

O
C O NH 2
OH O O O H
H N
N H H
C H 3- ( C H 2) 9 N N N O
H H O S
O O O O
C O NH 2
70
71

Fig. (5). Examples of peptidomimetic calpain inhibitors with various N-terminal capping groups.

Chloromethyl ketone 74 was described as a weak calpain and the kinetics of inhibition of most of the
irreversible nonpeptide inhibitor of calpain [138]. nonpeptide inhibitors are yet to be deciphered. It is
Isocoumarin analogues such as 75 are good conceivable that the compounds may inhibit calpain
inhibitors of serine proteases but are weak by binding to an allosteric site on the enzyme.
inhibitors of calpain (IC50 of 10 µM) [139].
Clavam-3-carboxamide derivatives such as 76
(23,500 M-1s-1) are irreversible inhibitors of D. Selectivity of Calpain Inhibitors
calpain [140]. Aurintricarboxylic acid 77 was Most peptidyl calpain inhibitors lack selectivity
demonstrated by Posner et al. [141] as a weak (IC50 for the enzyme. This lack of selectivity is inherent
of 22 µM) nonselective cell permeable calpain in the design of the inhibitors since the warheads of
inhibitor. Quinolinecarboxamides including 78 calpain inhibitors are identical to those found in
(IC50 of 510 nM) are fairly potent noncompetitive other proteases inhibitors. In the design of calpain
inhibitors of calpain [142]. Nonpeptide calpain inhibitors it was envisioned that appending general
inhibitors such as phevalin 4 (41), diketopiperazine protease inhibitor warheads to calpain preferring
5 (42), damnacanthal 7 (43) and penicillide 8 (44), subsite residues would generate selective inhibitors
which are obtained from natural sources were of the enzyme. However, this approach to selective
discussed earlier. The site of interaction with calpain inhibitors has enjoyed limited success
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1183

HO O OC H3
O OH
HN CH 3 CH O
O
N
N CH 3 OH

N O
O
4 5 7

O O H
N
H 3C O
CH 3 I SH
SH

O C O OH
HO C O OH F
HO
72 73
8

O
H
N N Cl
N
H
O
H
N O O
H2N O
O NH
N N (C H2 )6
O H
O S NH 2

Cl
74 75 76

O
O
C O OH
C O NH 2

N
H OO C C O OH
N Cl

HO OH
77 78
OH

Fig. (6). Examples of nonpeptide calpain inhibitors.

especially regarding selective inhibition of calpain calpain selectivity of dipeptide ketoamides by using
over cathepsin L. Leu-Phe or Leu-Abu at the P2-P1 position.
Chatterjee et al. [73] observed that incorporation Calpain is unique among the cysteine protease
of a xanthine group at the P3 position of family of enzymes in that it possesses a calcium-
ketomethylene calpain inhibitors such as 62 binding domain. Inhibition of calpain via
increased selectivity for calpain over cathepsin B by interaction of compounds with this domain
about 17-fold. In their study of a series of α-keto presents an attractive route to calpain selective
carbonyl containing calpain inhibitors Li et al. [75] agents. Indeed, Wang et al. [3, 136] have disclosed
noted that a number of the inhibitors were equally α-mercaptoacrylates (e.g. 72 and 73) as the first
potent with the calpains but some were an order of calpain inhibitors that inhibit the enzyme by binding
magnitude less effective with cathepsin B (Table 5). to the calcium domain. The compounds are highly
These investigators found that they could increase selective for calpain versus cathepsin B (Table 5).
1184 Current Medicinal Chemistry, 2000, Vol. 7, No. 12 I. O. Donkor

Table 5. Examples of Selective Inhibitors of Calpain

µM)
Ki (µ

Compounda Calpainb Cat. B (SR)c Ref.

Z-Leu-Abu-CONH(CH2)8CH3 0.12 150 (1,250) [75]

Z-Leu-Phe-COOH 0.0085 4.5 (529) [75]
Z-Leu-Phe-COOH 0.0057 (Cal. II) 4.5 (790) [75]
Z-Leu-Phe-COOEt 1.8 3.40 (189) [75]
Z-Leu-Phe-COOEt 0.40 (Cal. II) 3.40 (850) [75]
Z Leu-Phe-CONH(CH2)2Ph 0.052 9.3 (179) [75]
Z Leu-Phe-CONH(CH2)2Ph 0.024 (Cal. II) 9.3 (388) [75]
72 0.21 127.8 (609) [136]
73 0.26 >200 (>769) [136]
78 0.5 25 (50) [142]

Rate (M-1s-1)

t-Boc-Leu-Phe(DL)-CH2F 68,600 150 (457) [94]

Z-(D)-Ala-Leu-Phe-CH2-DFB 31,000 100 (310) [97]
Z-Leu-Leu-Phe-CH2S+(CH3)2Br- 200,000 (Cal. II)b 3,500 (57) [101]
Z-Leu-Leu-Tyr-CHN2 230,000 (Cal. II) b
1,300 (177) [143]
a
Z = Benzyloxycarbonyl; DFB =2,6-Di-fluorobenzene.
b
Calpain I was used except where indicated as calpain II.
c
Selectivity Ratios equal to or greater than 50 are reported.

Quinazolinecarboxamides such as 78 also show Leu-Leu-Phe-CH2 OCO[2,6-(CF3 )2 ]Ph, 1000 M -

over 50-fold selectivity for calpain over both s ) are poor inhibitors of calpain. The
1 -1

cathepsin B and cathepsin L. The compounds are difference in calpain inhibitory activity between
not active site directed inhibitors of calpain and these compounds was attributed to the steric bulk
hence may inhibit the enzyme by binding to a yet to of the arylacyloxy leaving group, which cannot be
be identified allosteric site [142]. tolerated by the active site of calpain. Crawford et
al. [143] showed that Z-Leu-Leu-Tyr-CHN2 is over
Chatterjee and coworkers [94] demonstrated that 170-fold selective for calpain II versus cathepsin B
t-Boc-Leu-Phe(DL)-CH2F is over 450-fold and but the compound is about 7-fold, a better inhibitor
680-fold selective for calpain I over cathepsins B of cathepsin L than calpain II.
and L, respectively. Harris and others [97] studied
a series of dipeptide and tripeptide
arylacyloxymethyl ketones and found that none of III. CONCLUSION
the dipeptides were selective for calpain versus
cathepsin L but a tripeptide with a P3 D-Ala residue A variety of peptidyl calpain inhibitors derived
was over 100-fold selective for calpain over from mammalian, plant, and synthetic sources have
cathepsins B and L. Pliura and coworkers [101] appeared in the literature. Majority of these
demonstrated that peptidyl sulphonium methyl inhibitors are active site directed reversible or
ketones (e.g. Z-Leu-Leu-Phe-CH2S+(CH3)2.Br-, irreversible agents with limited selectivity for
200,000 M-1s-1) are selective inhibitors of calpain calpain over other cysteine proteases. Most of the
over cathepsin B (3500 M-1s-1). Unlike sulfonium SAR studies undertaken to enhance the potency and
methyl ketones, acyloxymethyl ketones (e.g. Z- selectivity of the inhibitors involved modifications
Calpain Inhibitors Current Medicinal Chemistry, 2000, Vol. 7, No. 12 1185

to enhance binding to the unprimed subsite of [12] Otto, H-H.; Schirmeister, T. Chem. Rev. 1997, 97,
calpain. Though potency enhancement has been 133-171.
realized to some degree, selectivity via this
[13] Murachi T. Trends Biochem. Sci. 1983, 8, 167-169.
approach still remains a formidable challenge.
Compared to the unprimed subsite, the primed [14] Saido, T.; Sorimachi, H.; Suzuki, K. FASEB J.
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Future SAR studies should therefore concentrate
efforts at investigating the primed subsite of the [15] Bartus, R.T. Neurocientist 1997, 3, 314-327.
enzyme. The calcium-binding domain also affords [16] Murachi, T. Intracellular Ca2+ protease and its
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[18] Mehdi, S. Trends Biochem. Sci. 1991, 16, 150-153.

IV. ACKNOWLEDGEMENT
[19] Wang, K.K.W. Trends Pharm. Sci. 1990, 11, 139-
The author thanks NHLBI for grant 1 K14 142.
HL03536 to develop selective calpain inhibitors,
which provided the impetus to write this review, [20] Wang, K.K.W.; Yuen, P.-W. Trends Pharm. Sci.
article. 1994, 15, 412-419.

[21] Wang, K.K.W.; Yuen, P.-W. Adv. Pharm. 1997,

37, 117-152.
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