Cocrystal structures of primed side-extending α-ketoamide inhibitors reveal novel calpain-inhibitor aromatic interactions

Article abstract of DOI:10.1021/jm800045t

Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca²⁺ signaling. When Ca²⁺ homeostasis is disrupted, calpain overactivation causes unregulated proteolysis, which can contribute to diseases such as postischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (μI-II) bound to two α-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor toward μI-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.

Full text of DOI:10.1021/jm800045t

5264
J. Med. Chem. 2008, 51, 5264–5270
Cocrystal Structures of Primed Side-Extending r-Ketoamide Inhibitors Reveal Novel
Calpain-Inhibitor Aromatic Interactions#
Jin Qian,† Dominic Cuerrier,†,‡ Peter L. Davies,† Zhaozhao Li,§ James C. Powers,§ and Robert L. Campbell*,†
Department of Biochemistry, Queen’s UniVersity, Kingston, Ontario, K7L 3N6, Canada, and School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, GA 30332-0400
ReceiVed January 17, 2008
Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to
Ca2+ signaling. When Ca2+ homeostasis is disrupted, calpain overactivation causes unregulated proteolysis,
which can contribute to diseases such as postischemic injury and cataract formation. Potent calpain inhibitors
exist, but of these many cross-react with other cysteine proteases and will need modification to specifically
target calpain. Here, we present crystal structures of rat calpain 1 protease core (µI-II) bound to two
R-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected
aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain.
This interaction increased the potency of the inhibitor toward µI-II and heterodimeric m-calpain. Moreover,
stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address
regions, which could be incorporated into future inhibitors to enhance their calpain specificity.
In response to Ca2+ signaling, the calpain family of intra-
cellular cysteine proteases catalyzes the limited cleavage of
target proteins, resulting in changes to processes such as gene
expression, cytoskeleton remodeling, and apoptosis.1 Problems
arise following ischemic or cerebral injury, when cells lose their
ability to regulate Ca2+ influx to the cytoplasm. The elevated
Ca2+ concentration leads to calpain hyperactivation, which
causes uncontrolled proteolysis and irreversible cell damage.
Since their overactivation has been linked to the development
of pathological conditions such as stroke, Alzheimer disease,
Duchenne muscular dystrophy, and cataractogenesis, calpains
represent an important class of targets for pharmacological
inhibition.2,3
To date, all known calpain isoforms are multidomain en-
zymes,4 with a catalytic cleft located at the interface between
domains I and II.5 These two domains, which encompass the
enzyme’s proteolytic core, must each bind one Ca2+ ion to
facilitate the rearrangement of the catalytic triad and substrate
binding pocket into an active conformation.6 Although the
various other domains also contribute somewhat to calpain
activation, the susceptibility of full-length calpain to autolysis,
subunit dissociation, and aggregation following Ca2+ activation
has complicated its study in the full-length form.7 The protease
core, though, remains resistant to autolysis and maintains its
Ca2+-dependent activity, albeit at a significantly reduced level.8
In addition, because of the relative ease with which they can
be expressed in E. coli and crystallized, these protease cores
have become an invaluable tool for the structure-based design
of calpain inhibitors.9 While two structures have been reported
for the Ca2+-activated human protease core,10,11 in our hands,
the rat protease core has been much easier to purify and
crystallize. The sequences for the protease cores of rat and
human calpains 1 and 2 show a high degree of identity (87%
between rat and human calpains 1 and 70% between rat calpain
1 and human calpain 2). Furthermore, because the active site
clefts are particularly well conserved, the rat calpain 1 structure
remains a suitable model for designing and studying inhibitors
of calpain.
Of the reversible inhibitors that have been developed to target
calpains, most are peptide analogues containing an electrophilic
warhead group to covalently modify calpain’s active site
thiol.9,12,13 Although aldehyde and R-ketoamide functional
groups have been widely used as warheads, the latter has
emerged as the superior form with respect to both metabolic
stability and cell permeability.12 However, the poor specificity
of R-ketoamide inhibitors continues to limit their applicability
as potential therapeutic agents.2 Consequently, there has been
an increasing focus on designing peptidyl “address regions”
flanking the warhead to target the inhibitor to the calpain active
site. To improve specificity, these “address regions” are designed
to correspond with calpain’s residue preferences at each position
in a peptide substrate. For instance, µ-calpain’s protease core
(µI-II) demonstrates a preference for hydrophobic residues on
the N-terminal (unprimed) side of the scissile bond,14 specifically
phenylalanine and leucine at the P1 and P2 positions, respec-
tively. The crystal structure of µI-II in complex with 3 (SNJ-
1945),15 a peptidyl R-ketoamide containing this optimized
selection, shows each of the two side chains interacting with
the substrate binding cleft, thus showing how this unprimed
“address region” can target the warhead to calpain’s active site.
By itself though, this unprimed “address region” is insufficient
to confer specificity toward calpain, since the P2 leucyl side
chain is also accommodated by a hydrophobic pocket in other
cysteine proteases.16 Hence, there is an advantage to developing
an additional optimal “address region” on the C-terminal
(primed) side of the warhead. If the “address regions” on both
the unprimed and primed sides can be incorporated into a single
inhibitor, it would possess a substantially improved ability to
specifically target calpain.
#
Coordinates and structure factors for the complexes of the protease
core of µ-calpain with 1 and 2 have been deposited with the Protein Data
Bank (2R9C and 2R9F, respectively).
* To whom correspondence should be addressed. Phone: 613-533-6821.
Fax: 613-533-2497. E-mail: robert.campbell@queensu.ca.
†
Queen’s University.
Current address: Cytochroma Inc., 330 Cochrane Drive, Markham,
‡
Ontario, Canada L3R 8E4.
§
Georgia Institute of Technology.
10.1021/jm800045t CCC: $40.75
2008 American Chemical Society
Published on Web 08/15/2008

Calpain Inhibitor Structures
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5265
Figure 1. Chemical structures of 1 and 2, the two R-ketoamide calpain
inhibitors used in this study. Both compounds contain a P1 R-ami-
nobutanoic acid and P2 leucine residue, but the primed side extension
of 1 ends with adenine ring, while that of 2 is terminated by a piperazine
ring. The structure of the commercially available calpain inhibitor AK-
295 is also shown for comparison.
Previous studies on calpain inhibitors have shown that the
extension of an inhibitor into the primed region can increase
the inhibitor potency. For instance, Li et al.17 showed that the
inclusion of an arylalkyl primed-side substituent often improved
the potency toward both calpains 1 and 2 as well as cathepsin
B. In particular, the best primed-side substituent on the R-keto
amide was a (CH2)3-phenyl group. In another study, Chattergee
et al.18 examined the effect of different primed-side extending
biaryl sulfonamides and found that the inclusion of a spacer
between the two aryl motifs reduced the inhibitory activity.
Donkor et al.19 studied a series of inhibitors containing polar
substituents in the P1′ position within the linker to a phenyl
group. They modeled the polar group as interacting with Glu261,
which we later showed to reside in a flexible loop.15 The
common feature among these reports, though, is that an aromatic
moiety within the primed side was potentially beneficial to
inhibitory activity. However, without crystal structures of these
inhibitors bound to calpain, it is difficult to identify the
interactions that these primed-side substituents might make with
the active site.
Figure 2. Inhibitory effect of 1 and 2 on m-calpain (A) and µI-II (B).
Duplicate assays were performed by adding 1.3 µM (EDANS)-
EPLFAERK-(DABCYL) (1), 125 nM calpain (2), and 4 mM CaCl2
(3), then averaging to yield the plots shown. Autolytic inactivation of
m-calpain, but not µI-II, was observed when no inhibitor was added
(X). Immediately following addition of each inhibitor (4), the increase
in fluorescence intensity was attenuated. 1 (Y) caused a more distinct
attenuation of fluorescence in both the m-calpain and µI-II assays,
indicating that it is a more potent inhibitor when compared to 2 (Z).
developing more specific inhibitors that can simultaneously
target both the unprimed and primed subsites of calpains.
Results
Evaluating the Inhibitory Potency of 1 and 2 on µI-II
and m-Calpain. Upon calcium activation, the rate of cleavage
of the FRET substrate by m-calpain (Figure 2A) was ap-
proximately 4 times higher than that catalyzed by µI-II (Figure
2B). In the absence of either inhibitor, the autolytic inactivation
of m-calpain caused the fluorescence intensity to plateau after
just a few minutes of reaction. However, this autolytic inactiva-
tion was not observed with µI-II. Upon addition of either
inhibitor, the increase in fluorescence was immediately attenu-
ated, albeit more noticeably with m-calpain. For both m-calpain
and µI-II, 1 caused a more distinct attenuation of fluorescence
when compared to 2, indicating that the former is a more potent
calpain inhibitor.
X-ray Crystal Structures of the Complexes of µI-II
with 1 and 2. Owing to its stability and resistance to autolytic
inactivation,8 µI-II was used to form calpain-inhibitor com-
plexes with 1 and 2. A summary of crystallographic data
collection and refinement statistics is presented in Table 1.
Within the catalytic cleft of µI-II, the electron density of each
inhibitor was clearly observed in the inner S-unprimed and
Here, we present the crystal structures of µI-II bound to two
R-ketoamide inhibitors 1 and 2 (Figure 1), each possessing
substituents that extend deeper into the µI-II primed side cleft
than previously observed. These structures also show the
movement of a flexible gating loop out of the binding pocket
in the presence of a ligand moiety stretching into the S-primed
subsites. More importantly, the structure of µI-II bound to 1
demonstrates a novel noncovalent interaction between µI-II and
the inhibitor, where the aromatic adenine moiety of 1 stacks
against a conserved tryptophan in the catalytic cleft of µI-II
and forms a hydrogen bond to the side chain of Glu300. These
interactions may explain why 1 inhibits µI-II activity more
potently than the nearly identical compound 2, which possesses
a nonaromatic primed side substituent. The information gained
from these structures should provide valuable insights for

5266 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Table 1. Summary from Data Collection and Structure Refinement
Qian et al.
three-carbon chain that lies on the C-terminal side of the
warhead and is ultimately transmitted to the terminal ring groups
(Figure 4A). It appears that the primed-side three-carbon chain
in 1 may adopt a different conformation from that of 2 in order
to facilitate an aromatic stacking interaction between the adenine
moiety of 1 and the side chain of Trp298. As shown in Figure
4B, it stacks in a coplanar conformation with respect to
tryptophan’s indole group, leaving a gap of only 3.5 Å between
the two and allowing the formation of a hydrogen bond to
Glu300. Neither this coplanar stacking nor any polar contacts
were observed between the nonaromatic piperazyl ring of 2 and
the primed side pocket (Figure 4C). The absence of this stacking
interaction and a hydrogen bond to Glu300 therefore likely
accounts for the weaker electron density around the piperazyl
ring when compared to that of the adenine moiety (parts A and
B of Figure 3).
For both inhibitor-bound structures, although the electron
density of µI-II was strong in most areas, the density was
observed to be considerably weaker in residues 251-261, which
comprise a known flexible loop region. Fitting these residues
to the available density resulted in a loop that adopted an “open”
conformation, with Glu261 displaced away from the S1′ subsite.
This “open” conformation closely resembles the one reported
in the structure of 3 bound to µI-II but contrasts with the
“closed” conformation observed in the leupeptin-bound structure
(Figure 5).
µI-II-1 complex
Crystal Parameters
µI-II-2 complex
space group
a (Å)
P212121
40.43
P212121
40.57
b (Å)
70.24
70.57
c (Å)
110.41
110.41
Data Collection Statistics
resolution limit (Å)
1.80-59.23
(1.80-1.90)
196538 (28320)
29796 (4224)
99.2 (98.4)
35.1 (15.1)
4.0 (12.2)
1.60-35.29
(1.60-1.69)
229854 (15208)
40801 (4932)
96.0 (82.6)
17.9 (1.6)
no. of measured reflections
no. of unique reflections
completeness (%)
I/σ(I)
Rmerge (%)
7.8 (60.2)
Refinement Statistics
resolution range (Å)
1.80-27.22
1.60-35.29
(1.60-1.642)
5 (5)
18.9 (33.8)
22.2 (34.7)
2663/268/2/3/12
(1.80-1.847)
size of free reflection set (%)
Rwork
Rfree
no. of atoms (no H), protein/
solvent/Ca2+/Cl-/glycerol
bond angle rmsd (deg)
bond length rmsd (Å)
average B-factor (Å2)
B-factor from Wilson
plot (Å2)
5 (5)
15.6 (16.9)
19.6 (24.1)
2644/305/2/3/12
1.47
0.016
14.3
13.0
1.273
0.012
15.8
17.5
S-primed subsites (parts A and B of Figure 3). Electron density
was weakest around the P3 phenyl ring of both inhibitors,
suggesting that this group was more flexible and formed limited
interactions with the S3-unprimed region. Nevertheless, when
compared to the previously solved structures of µI-II bound to
leupeptin or the R-ketoamide inhibitor 3,15 the position of the
P3 phenyl ring in 1 and 2 was consistent with that of the P3
diethylene glycol in 3 and the P3 leucyl group in leupeptin; all
four P3 groups overlap closely along the surface of domain I
(Figure 4A).
Both inhibitors 1 and 2 are stabilized within the active site
through interactions that are conserved in the structures of µI-
II bound to leupeptin or 3. On the unprimed side, hydrogen
bonds are formed between the inhibitor’s peptide backbone
atoms (amide nitrogen and carbonyl oxygen of the P2 residue
and amide nitrogen of the P1 residue) and Gly208 and Gly271
in the catalytic cleft (parts C and D of Figure 3). At the
R-ketoamide warhead of 1, 2, and 3, the newly formed hydroxyl
group, which arises from nucleophilic attack by the active site
thiol, forms a hydrogen bond with the imidazole ring of His272,
while the carbonyl oxygen of the carboxyamide provides further
stabilization via two polar contacts with Gln109 and Cys115.
As would be expected from the highly conserved hydrogen
bonding interactions, a high degree of structural alignment was
observed at the P2 leucine of each of the inhibitors 1, 2, 3 and
leupeptin (Figure 4A). In fact, the backbone and side chains
adopt nearly identical conformations. This strong overlap
continued into the backbone of the P1 residue and its respective
side chain ꢀ- and γ-carbons. In addition to the conserved
hydrogen bonds, another contributing factor to the high degree
of structural alignment is the narrowness of the binding pocket
around the P1 residue. As described by Cuerrier et al.,15 this
narrow pocket imposes restrictions on the ability of the P1
residue to rotate around the CR-Cꢀ bond.
Discussion
As noted in previously solved µI-II structures, residues
251-261 in domain II comprise a flexible loop that possesses
weak electron density and is considered to act as a gate for the
catalytic cleft.11,15,20 When µI-II is bound to an inhibitor that
extends only into the unprimed side, such as leupeptin, the
flexible “gating” loop adopts the “closed” conformation, with
Glu261 obstructing the S1′ subsite (Figure 5). However, in the
presence of an inhibitor such as 1 or 2, which possesses a primed
side group that extends into the S2′ subsite, the loop is oriented
away from the active site in a much more “open” manner. This
“open” conformation was first observed in the structure of µI-
II bound to 3, where the inhibitor’s P1′ cyclopropyl group was
thought to displace Glu261 from the S1′ site.15 Although the
purpose of the “open” and “closed” loop conformations has yet
to be determined, it is possible that they could be signaling
different catalytic stages, possibly via interactions with domains
(e.g., domain III) that are absent in the isolated proteolytic core.
The open loop conformation, for instance, might be signaling
the initial substrate-binding event where the polypeptide chain
would stretch across the cleft. However, once catalysis has
yielded the acyl-enzyme intermediate, which no longer possesses
a primed side extension, the loop may close to signify the near
completion of catalysis.
In addition to providing insight about the flexible gating loop,
the crystal structures produced from this study also revealed
the importance of the newly discovered aromatic stacking
interaction between the primed-side adenine moiety of 1 and
the indole ring of Trp298. This class of noncovalent interactions,
commonly referred to as π-π stacking, has been widely
documented in protein-ligand complexes, especially when the
ligand comprises nucleic acids.21-26 One study in particular
examined the stacking interactions between adenine and aro-
matic residues in the active site of DNA glycosylases.27 Since
these enzymes remove damaged bases during the first step of
base excision repair28 and have aromatic residues lining their
active sites, it was thought that π-π stacking was responsible
While the structures of leupeptin and 3 are terminated at, or
shortly after, the warhead, 1 and 2 possess much longer primed
side extensions. Interestingly, the greatest degree of structural
divergence between these two inhibitors is concentrated within
this region. Structural alignment deteriorates substantially in the

Calpain Inhibitor Structures
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5267
Figure 3. Crystal structures of 1 and 2 bound to the active site of µI-II. The electron density of 1 (calculated with coefficients 2mFobs - DFcalc and
ꢁ
calc and contoured at 1σ) and the inhibitor’s polar contacts with µI-II are shown in parts A and C, respectively, while those for 2 are shown in parts
B and D. The “carve” feature of PyMOL33 was used to limit the display of electron density to a distance of 2 Å from the inhibitors. Bonds are
colored by atom type: carbon ) gray (µI-II), carbon ) green (1), carbon ) cyan (2), nitrogen ) blue, oxygen ) red, sulfur ) orange. The peptide
subsites are labeled below part C.
for substrate recognition and stabilization. By examination of
the available crystal structures of DNA glycosylases and
performance of computational analyses of the aromatic interac-
tions, it was determined that out of the four aromatic amino
acids (His, Phe, Tyr, and Trp), the strongest stacking with
adenine occurred in the presence of tryptophan, an apparent
consequence of its side chain’s large surface area and dipole
moment.27 Furthermore, the optimal perpendicular separation
between tryptophan and adenine was determined to range from
3.5 to 3.6 Å, which matches the distance observed in the
complex of µI-II with 1.
On the basis of the energetic consequences of π-π stacking
and considering that compounds 1 and 2 differ only with respect
to their terminal primed side ring groups, the aromatic interaction
therefore may explain the greater inhibitory potency of 1 over
2 observed in the fluorimetry assays (Figure 2). Through this
stacking, the terminal primed side end of 1 is held more rigidly
in place and likely works in conjunction with the unprimed side
polar contacts to properly orient the R-keto carbonyl relative to
the active site thiol (Figure 3). In contrast, the absence of this
aromatic interaction in the structure of µI-II with inhibitor 2
causes the piperazyl ring to exhibit greater flexibility, which
could be transmitted toward the warhead group, thereby
impairing the R-keto carbonyl from achieving the necessary
orientation required for potent inhibition.
into the orientation observed in the inhibitor-bound structures.
In this position, it helps shield two of the three catalytic triad
residues (His272 and Asn296) from solvent exposure and thus
facilitates catalysis. Therefore, the π-π stacking is done by
Trp298 in its catalytically active conformation, suggesting that
inhibitor 1 binds preferentially to the Ca2+-bound, active form
of calpain.
Although the π-π stacking interaction on the primed side
cleft is promising from the perspective of structure-guided drug
design, it alone is expected to be insufficient to specifically target
calpains, since other cysteine proteases also possess a tryptophan
residue that resides in a position similar to that in the catalytic
conformation of calpain.6 As shown in Figure 6, when the
structures of the leupeptin-bound papain, human liver cathepsin
B, and E-64-bound cathepsin K are aligned with that of the
µI-II-1 complex, the analogous tryptophan residue in these
other cysteine proteases could still interact with the adenine ring
of 1, albeit perhaps less optimally when compared to Trp298.
What the other papain-like cysteine proteases lack, though, is a
residue equivalent to Glu300, so they would be unable to form
a hydrogen bond to the amino group of the adenyl moiety. Along
these lines, the stacked adenine ring could still be used as a
scaffold for other functional groups, which could form contacts
with structural features that are unique to calpain’s primed side
cleft. For instance, the shallow hydrophobic pocket formed by
Ala262, Ile263, and Val269 (Figure 4A) is not observed in the
primed side cleft of papain, cathepsin B, or cathepsin K.
Therefore, addition of an aliphatic extension to the adenine ring
could facilitate interactions with the hydrophobic pocket. In
addition, the replacement of the primary amino group with a
positively charged substituent on the adenine moiety of 1 could
Incidentally, the residue that facilitates the π-π stacking
interaction, Trp298, is also involved in the later stage of active
site assembly, where its side chain undergoes a radical change
in position.6 Upon binding of the second and final Ca2+ ion to
the µ-calpain protease core, the indole ring of Trp298 moves
from its initial wedgelike position between the catalytic domains

5268 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Qian et al.
Figure 5. Overlay of four µI-II structures, showing the two possible
conformations of the gating loop (residues 256-261). Glu261 in µI-II
and the bound inhibitor 1 are shown as sticks. The primed side
extensions of 1 and 2 cause Glu261 in the gating loop to be displaced,
resulting in an “open” conformation. This “open” conformation closely
resembles that of the structure of µI-II bound to 3, where a P1′-
cyclopropyl group causes Glu261 to be displaced. When bound to
leupeptin, which lacks a primed side extension, the gating loop is in a
“closed” position, with Glu261 blocking the S1′ subsite. The polypep-
tides are colored as follows: green ) µI-II-1 complex, cyan ) µI-
II-2 complex, magenta ) µI-II-3 complex, yellow ) µI-II-leupeptin
complex.
Figure 4. (A) Structural overlap of calpain inhibitors, achieved by
aligning the polypeptide chain of each structure. Inhibitors are shown
as sticks and accompanied by a surface representation of µI-II from
the µI-II-1 complex. Atoms are colored as in Figure 3. The overlap
of 1, 2, 3, and leupeptin shows a high degree of structural alignment
from the P3 to the P1 residue. The hydrophobic pocket formed by
Ala262, Ile263, and Val269 is highlighted in light green. (B, C)
Orientation of terminal primed side ring groups of 1 and 2 relative to
Trp298 in µI-II. A transparent surface representation of µI-II is also
shown. (B) Primed side aromatic stacking interaction between the
adenine moiety of 1 and W298. (C) This stacking interaction is absent
for the nonaromatic piperazyl group of 2. Atoms are colored as for
Figure 3.
Figure 6. Overlay of Trp298 in the µI-II-1 complex (µI-II in white,
1 in green) with the corresponding tryptophan in papain (magenta),
cathepsin B (yellow), and cathepsin K (cyan). This overlay was achieved
by first aligning three catalytic site residues (cysteine, histidine,
tryptophan) of papain, cathepsin B, and cathepsin K with those of the
µI-II-1 structure. Inhibitor 1 and the overlaid cysteine, histidine, and
tryptophan residues are shown as sticks. The relatively similar
alignments between the tryptophans suggest that aromatic stacking, by
itself, will be insufficient to specifically target an inhibitor into the
calpain active site. The PDB entries for the cysteine proteases are as
follows: papain bound to leupeptin (1POP), human liver cathepsin B
(1HUC), and cathepsin K bound to E-64 (1ATK). Nitrogens are colored
blue, while oxygens are red.
enhance the electrostatic contacts with the Glu300 side chain.
Modification of compound 1 to exploit these calpain-exclusive
interactions holds promise for the development of new potent
and selective inhibitors.
In conclusion, we have demonstrated that interactions with
calpain’s S-primed subsites represent an important aspect of
inhibitor design that should not be ignored. As was highlighted
in the two crystal structures, the aromatic stacking interaction
and hydrogen bond between the primed side adenine ring of 1
and Trp298 and Glu300, respectively, of µI-II, provided the
inhibitor with a decisive potency advantage over an equivalent
inhibitor lacking a terminal aromatic group. Being the first
noncovalent interactions reported on the primed side of a
calpain-inhibitor complex, the two high-resolution structures
described in this study should contribute greatly to the field of
structure-guided drug design and in particular to the develop-
ment of potent and specific inhibitors of calpains.
Experimental Procedures
Materials. The rat µI-II construct is composed of Gly27-Asp356
from rat µ-calpain and contains an N-terminal Met and C-terminal
Leu-Glu-(His)6 tag. The full-length rat m-calpain, comprising the
80 and 21 kDa subunits, contains the C-terminal Lys-Leu-Ala-Ala-
Leu-Glu-(His)6 tag. Both constructs were recombinantly expressed
in E. coli and purified as previously described.8,29 The calpain
substrate (EDANS)-EPLFAERK-(DABCYL) was synthesized at the

Calpain Inhibitor Structures
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5269
Alberta Peptide Institute (Edmonton, Alberta, Canada) and in the
Peptide Synthesis Laboratory of the Protein Function Discovery
Facility at Queen’s University (Kingston, Ontario, Canada). All
other reagents were obtained from common sources.
Synthesis of the r-Ketoamide Compounds. 3-(Benzyloxycar-
bonyl-L-leucylamino)-N-(3-(6-amino-9H-purin-9-yl)propyl)-2-oxo-
pentanamide (1, Z-Leu-Abu-CONH-(CH2)3-adenin-9-yl). A mixture
of adenine (4.05 g, 30 mM), 1-bromo-3-chloropropane (21.3 g, 13.4
mM), and potassium carbonate (10.4 g, 75 mM) in DMF (200 mL)
was stirred at room temperature under argon for 4 days, then filtered
and evaporated to dryness. The crude product was washed with
water and dried. Recrystallization from ethanol gave 9-(3-chloro-
propyl)adenine in 59% yield.
9H, CH3 of Val and Abu), 1.60-1.80 (m, 5H, CH2 and CH), 2.00
(m, 2H, CH2), 2.44 (s, 3H, CH3 of piperazine), 2.50-2.65 (m, 8H,
CH2 of piperazine), 3.30 (m, 2H, CH2), 4.20 (m, 3H, CH2 and R-H),
5.10 (s, 2H, Z), 5.15 (m, 1H, R -H), 6.70 (b, 1H, NH), 7.20-7.30
(m, 6H, Ph and NH), 8.60 (b, 1H, NH).
Fluorimetric Analysis of µI-II and m-Calpain Inhibition
by 1 and 2. Enzymatic activity was monitored in real time using
a 1.5 mL quartz cuvette and a Perkin-Elmer LS50-B luminescence
spectrophotometer. Duplicate assays were performed with 1.3 µM
(EDANS)-EPLFAERK-(DABCYL)12 and 125 nM µI-II or m-
calpain in a buffer solution containing 50 mM HEPES-HCl (pH
7.7) and 10 mM DTT. The reaction was initiated at the 420 s time
mark using 4 mM CaCl2 and monitored at excitation and emission
wavelengths of 335 and 500 nm, respectively. After a waiting period
of 100 s, the inhibitor solution (either 1 or 2 dissolved in DMSO)
was added at a concentration of 1 µM (for the m-calpain assays)
or 50 µM (for the µI-II assays). Control assays were performed by
adding an equivalent volume of DMSO after the 100 s delay.
Determination of µI-II-1 and µI-II-2 Crystal Structures.
A solution of 325 µM µI-II in 10 mM HEPES-HCl (pH 7.7) and
10 mM DTT was first incubated with 2 mM 1 or 2 at room
temperature for 30 min. This solution was then mixed with an equal
volume of a precipitant solution to cover conditions that expanded
around 1.5-2.0 M NaCl, 10 mM CaCl2, and 0.1 M MES (pH
5.75-6.5). By use of the hanging drop vapor diffusion method,
crystals were obtained and subsequently soaked in a solution of
the mother liquor supplemented with 20% glycerol, prior to data
collection. Crystallographic data were collected at beamline X6A
at the National Synchrotron Light Source, Brookhaven National
Laboratory. The processing of data was performed using Mosflm31
and Scala32 from the CCP4 program suite.33 The inhibitor-bound
structure was solved using the Ca2+-bound µI-II (PDB code 1KXR)
as the model for molecular replacement in MOLREP.34 The
molecular topology descriptions of the inhibitors 1 and 2 were
generated with the Dundee PRODRG2 server.35 Model refinement
and building were performed using REFMAC536 and Coot,37
respectively. The structures shown in Figures 3-6 were prepared
with PyMOL.38
The 9-(3-chloropropyl)adenine (1.9 g, 9 mM) and sodium azide
(1.75 g, 27 mM) in DMF was stirred at 80 °C for 24 h, cooled to
room temperature, and filtered. The solid was washed with CH2Cl2.
The solvent was removed from the combined filtrates, and the
residue was taken up in water with sonication. The aqueous layer
was extracted with CH2Cl2 (3 × 60 mL). After removal of the
solvent, the crude product was recrystallized from ethanol to give
9-(3-azidopropyl)adenine as a white crystalline solid in 81% yield.
The 9-(3-azidopropyl)adenine (0.5 g, 2.3 mM) and 5% palladium
on carbon (0.5 g) in methanol were reacted with hydrogen gas at
room temperature for 22 h. The catalyst was removed by filtration,
and the solvent was removed to give 9-(3-aminopropyl)adenine as
1
a white solid in 76% yield. H NMR (DMSO-d6): 1.80 (m, 2H,
CH2), 2.45 (m, 2H, CH2), 3.35 (s, 2H, NH2), 4.20 (m, 2H, CH2),
7.20 (s, 2H, NH2), 8.10 (s, 2H, CH). MS (ED+): 193.0.
The ketoamide product Z-Leu-Abu-CONH-(CH2)3-adenin-9-yl
was obtained from 9-(3-aminopropyl)adenine and the ketoacid
Z-Leu-Abu-COOH30 using the EDC/HOBt coupling method, puri-
fied by column chromatography on silica gel with 85:15 CH2Cl2/
MeOH as the eluant, then recrystallized from CH3COOEt/hexane
to give a white solid (27% yield). 1H NMR (CDCl3): 0.91 (m, 9H,
CH3 of Val and Abu), 1.60-1.80 (m, 5H, CH2 and CH of Leu and
Abu), 2.00 (m, 2H, CH2), 3.20 (2H, CH2), 4.24 (m, 3H, CH2 and
R-H), 5.11 (s, 2H, Z), 5.20 (m, 1H, R-H), 6.20 (s, 1H, NH), 6.80
(b, 1H, NH), 7.20-7.40 (m, 6H, Ph and NH), 7.85 (d, 1H, CH of
adenine), 8.36 (d, 1H, CH of adenine).
Acknowledgment. The authors thank Sherry Gauthier for
technical assistance and Marc Allaire from beamline X6A at
the National Synchrotron Light Source, Brookhaven National
Laboratory, for assisting with the crystallographic data collec-
tion. We thank Christina Hampton and Dr. Facundo Fernandez
in the School of Chemistry and Biochemistry at Georgia Institute
of Technology for the HPLC/MS data on the calpain inhibitors.
This work was funded by a grant from the Canadian Institute
for Health Research to P.L.D. and National Institutes of Health
(Grant R21 NS053801) to J.C.P. J.Q. is supported by a
studentship from the Government of Canada’s Natural Sciences
and Engineering Research Council. P.L.D. holds a Canada
Research Chair in Protein Engineering.
3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(4-methylpiperazin-
1-yl)propyl)-2-oxopentanamide (2, Z-Leu-Abu-CONH-(CH2)3-(4-
methylpiperazin-1-yl). A solution of N-(3-bromopropyl)phthalimide
(8.04 g, 30 mM) in xylene (60 mL) was added dropwise to a
solution of 1-methylpiperazine (6.61 g, 66 mM) in xylene (90 mL)
at 70 °C. After the addition was complete, the mixture was heated
under reflux for 20 h. The precipitate was removed by filtration,
and the filtrate was concentrated. The crude product was purified
by silica gel chromatography with 9:1 CH2Cl2/MeOH to give the
product N-(3-(4-methylpiperazin-1-yl)propyl)phthalimide as an oil
in 72% yield.
A solution of N-(3-(4-methylpiperazin-1-yl)propyl)phthalimide
(6.2 g, 21.6 mM) and hydrazine monohydrate (1.13 g, 26 mM) in
ethanol (60 mL) and methanol (60 mL) was refluxed for 4 h. After
the mixture was cooled to room temperature, concentrated HCl (2.4
mL) was added and the mixture heated under reflux for another
1 h. After removal of the solvent, water (100 mL) was added, the
mixture stirred, and insoluble material removed by filtration. Solid
K2CO3 (1.2 equiv) and CH2Cl2 (100 mL) was added to the aqueous
layer, and the mixture was stirred and filtered. The organic layer
was washed with water (3 × 20 mL). The combined aqueous layers
were washed with Et2O. Water was removed from the organic
layers. They were then dried and evaporated to give 1-(3-
Supporting Information Available: Analytical data (high
resolution mass determination and HPLC tracings) for target
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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