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21.2002.BP63 Ritonavir inhibition of calcium-activated neutral proteases. Authors: Wenshuai Wan, Paolo B. DePetrillo

21.2002.BP63 Ritonavir inhibition of calcium-activated neutral proteases. Authors: Wenshuai Wan, Paolo B. DePetrillo

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Short communication
Ritonavir inhibition of calcium-activated neutral proteases
Wenshuai Wan, Paolo B. DePetrillo
*
Unit of Clinical and Biochemical Pharmacology, Laboratory of Clinical Studies, Division of Intramural Clinical and Biochemical Research, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 10/3C103, 10 Center Drive MSC 1256, Bethesda, MD 20892-1256, USA
Received 13 August 2001; accepted 15 November 2001
Abstract
Calpains (EC 3.4.22.17) are intracellular calcium-activated cysteine proteases that mediate tissue injury following post-ischemic andpost-traumatic stress. Both human HIV protease and calpains share a similar secondary structure, where the active site is ¯anked byhydrophobicregions.Thepresent studydemonstrates thatritonavir,ahydrophobicHIVproteaseinhibitor,alsoinhibitscalpainactivity.InPC12 cell extracts assayed for calpain at maximal activity (2 mM calcium), ritonavir exhibited competitive inhibition with a
i
of 11
Æ
7
:
0
m
M. Experiments with puri®ed enzymes showed inhibition for both m- and
m
-calpain isoforms (m-calpain,
i
9
:
2
Æ
1
:
2
m
M;
m
-calpain,
i
5
:
9
Æ
1
:
4
m
M). Ritonavir also inhibited calcium-stimulated calpain activity in PC12 cells
in situ
. These results suggestthat ritonavir or analogues of the drug should be investigated as cytoprotective agents in conditions where cell death or injury is mediatedvia calpain activation.
#
2002 Elsevier Science Inc. All rights reserved.
Keywords:
Calpain; Enzyme inhibitors; Ritonavir; PC12 cell; HIV protease
1. Introduction
Calpains (EC3.4.22.17) exist as heterodimerscontaininga unique heavy 80-kDa subunit and a common 30-kDaregulatory subunit. At least three unique gene products of the 80-kDa subunit can be identi®ed in mammalian cellsbasedontheircalciumrequirementsforproteolyticactivity:
m
-calpain, requiring 5±50
m
M calcium; m-calpain, requir-ing 150±1000
m
M calcium; and p94, requiring intermediateconcentrations of calcium for half-maximal activity [1,2].Calpain inhibitors are neuroprotective in models of traumatic brain and myocardial injury [3±9]. Studies haveindicated that some calpain inhibitors exhibited protectiveeffects against neuronal damage both
in vitro
and
in vivo
[10±12]. These promising studies suggest that calpaininhibition may ®nd therapeutic applications in the treat-ment of post-ischemic or post-traumatic tissue injury [13].For both human HIV protease and calpains, the potencyof inhibitory compounds is increased by hydrophobicgroups ¯anking non-hydrolyzable pseudo-peptide bonds[14,15]. The active site in both proteases is also located inthe terminal pocket of a groove ¯anked by hydrophobicdomains [16,17]. While HIV protease is an aspartyl pro-tease,andcalpainsarecysteineproteases,peptidealdehydeinhibitors of calpain were shown previously to also inhibitHIV protease [18]. Taken together, these ®ndings sug-gested that ritonavir (Fig. 1), a hydrophobic HIV proteaseinhibitor, might also demonstrate activity as a calpaininhibitor.ThepresentstudyusedPC12cellextractstocharacterizethe mechanism of protease inhibition by ritonavir. Thekinetics ofinhibition were con®rmed using puri®ed m- and
m
-calpain preparations. These results were extended to awhole cell assay, based on the ability to monitor calpainactivation in live whole cells.
2. Materials and methods
2.1. PC12 whole cell preparation
Unless otherwise stated, all reagents were obtained fromtheSigma ChemicalCo. PC12 cells were culturedin RPMImedium (Gibco) containing 5% fetal bovine serum, 10%heat-inactivated horse serum, and 50 mg/L of gentamicinat 37
8
, 5% CO
2
. Cells were isolated by centrifugation(200
g
for 4 min at 25
8
) and suspended in Hanks' BalancedSalt Solution (HBSS). Enzymatic activity was measured aspreviously described for whole cell experiments [19].
Biochemical Pharmacology 63 (2002) 1481±14840006-2952/02/$ ± see front matter
#
2002 Elsevier Science Inc. All rights reserved.PII: S0006-2952(02)00907-3
*
Corresponding author. Tel.:
1-301-496-9420; fax:
1-301-402-0445.
E-mail address:
pbdp@helix.nih.gov (P.B. DePetrillo).
 
2.2. PC12 extract preparation
PC12 cells were isolated as described above, and cellpellets were suspended in 1 mL of extract buffer, contain-ing 30 mM Tris at pH 6.8, 15 mM EDTA, 5 mM EGTA,1 mM dithiothreitol, 100
m
g/mL of phenylmethylsulfonyl¯uoride, 0.1
m
g/mL of 4-(2-aminoethyl)benzenesulfonyl¯uoride (AEBSF), 14.4 mM 2-mercaptoethanol, and 1%Triton X-100. After one cycle of freeze±thaw, the suspen-sion was centrifuged at 14,000
g
for 20 min at 4
8
, andaliquots of the supernatant were used in the experiments.The protein concentration of the supernatant used for theassays was approximately 4 mg/mL.
2.3. m-Calpain- and 
m
-calpain-purified enzyme preparation
Calpains were suspended at a concentration of 2.6 units/ mL for m-calpain (Calbiochem-Novabiochem) and 0.53units/mL for
m
-calpain (Biovision Research) in a buffersolution containing 60 mM imidazole (pH 7.3), 5 mM
L
-cysteine (Fluka), and 2.5 mM 2-mercaptoethanol.
2.4. Calpain activity assays
Calpain activity in cell extracts was measured usingthe ¯uorescent calpain peptide substrate
-succinyl-Leu-Tyr 7-amido-4-methylcoumarin as previously described[19]. Fluorescence activity was monitored with a Spec-traMAX Gemini microplate spectrophotometer (Molecu-lar Devices) at 380 nm excitation and 480 nm emission.Foursubstrateconcentrations(0,80,240and720
m
M)andfour ritonavir (Moravek Biochemicals, Inc.) concentra-tions (0, 1, 10 and 100
m
M) were used for kinetic deter-minations. In assays using PC12 whole cells, the substrateconcentrationwasheldat80
m
M.Thereactionwasstartedby adding 10
m
L of extract or cell suspension to the wells.Inpuri®edenzymeexperiments,5
m
Lofbuffercontainingenzyme (m-calpain, 2.6 units/mL;
m
-calpain, 0.53 units/ mL) was added to the wells. Total volume for assay was150
m
L.For PC12 extract experiments, calpain speci®c activitywas obtained by subtracting the rate obtained in theabsence of calcium from the rate obtained in the presenceof calcium. For puri®ed enzyme experiments, no baseline¯uorescence was observed in the absence of added cal-cium.
2.5. Kinetic analysis and statistics
Data consisting of relative ¯uorescence units (RFU) wasobtainedevery 2 min for1 hr.Datawere analyzedbasedoninitial rate, de®ned as the slope of the increase of theobtainedRFU value uptoamaximumtimeof10 min.Datapoints from 7±9 separate experiments were used for thekinetic analyses. Each experimental point was determinedas the mean of six replicates. Maximal ratewas obtained inthe presence of 2 mM calcium. Steady-state kinetic datawere ®t to a series of model equations describing compe-titive, uncompetitive, and noncompetitive inhibition. Non-linear regression with an adaptive non-linear squaresalgorithm [20] was employed for the analyses utilizingNLREG software written by Phillip H. Sharrod. Modelsuf®ciency was evaluated based on parameter convergencewith a tolerance factor of 1
Â
10
À
10
,the overall F-value forthe regression, and the magnitude and sign of the para-meters obtained after convergence.To take into account differences in enzymatic activitybetween experiments, the kinetic parameters in experi-ments were estimated based on the ratio
:
v
i
v
m
(1)where
v
i
is the velocity in the presence of inhibitor, and
v
m
is the velocity in the absence of inhibitor. We chose thefollowingsetsofkineticequationsbasedontheassumptionthat ritonavir was a substrate analogue and would thereforeinhibit product formation [21]. This scheme is a modi®ca-tion of a previously described method useful in character-izing inhibitors following ¯uorescence-based studies of enzymatic activity [21].For a competitive inhibitor, the initial reaction velocityfrom steady-state kinetics is:
v
max
1
 
a
=
 A
 Â 
1
=
i

(2)where
v
initial velocity;
concentration of inhibitor;
i
 

 I 
=
EI 
;
A
substrate concentration; and
a
Michaelis±Menten constant. The rate in the absence of theinhibitor is:
v
m
max
1
a
=
 A
Wede®nearatio(
)betweentheobservedvelocityinthepresence of the inhibitor and the velocity in the absence of the inhibitor, where 0
<
1:
v
i
v
m
max
=
1
 
a
=
 A
 
1
=
i

max
=
1
a
=
 A
(3)
Fig. 1. Molecular structure of ritonavir. Hydrophobic planar groups areseen flanking non-hydrolyzable pseudo-peptide bonds.1482
W. Wan, P.B. DePetrillo/Biochemical Pharmacology 63 (2002) 1481±1484
 
For competitive, uncompetitive, and noncompetitiveinhibition, the kinetic equations therefore reduce to thefollowing:
A
a
 A
a
1
=
i
f
Competitiveinhibition
g
(4)
A
a
 A
1
=
i
 
a
f
Uncompetitiveinhibition
g
(5)
A
a
 A
1
=
ii
 
ia
1
=
i
f
Noncompetitiveinhibition
g
(6)
where
i
is the dissociation constant for the enzyme±inhibitor complex,
ia
is the dissociation constant of theenzyme±substrate complex, and
ii
is the dissociationconstant of the enzyme±substrate±inhibitor complex.
3. Results
When the kinetics of calpain activity inhibition byritonavir were examined in PC12 cell extracts, the bestmodel ®t was found with the equation describing compe-titive inhibition, having an overall F-value of 118.48 withan associated
P
<
0
:
00001. The observed
i
for ritonavirwas 11
Æ
7
:
0
m
M (mean
Æ
SEM). Noncompetitive anduncompetitive inhibition models were rejected becausethe parameters either failed to converge or converged tonegative values. Since only the competitive model wasfound to converge, it was not necessary to compare model®t taking into account the number of model parameters.With experiments employing cell extract, the observedkinetic parameters represent aggregate estimates sinceboth m- and
m
-calpain isoforms were present.Enzymatic studies were performed to characterize theinhibitory properties of both puri®ed isoforms of calpain.Ritonavir exhibited competitive inhibition with
i
9
:
2
Æ
1
:
2
m
M (F-value
353
:
81,
P
<
0
:
00001) against m-cal-pain. Ritonavir also exhibited competitive inhibition with
i
5
:
9
Æ
1
:
4
m
M (F-value
28
:
72,
P
0
:
0002) against
m
-calpain. Results for both sets of experimentsareshown inTable 1.Substrate hydrolysis in the presence of ritonavir wasexamined in whole PC12 cells in the presence of 10
m
Mionomycin and 1.4 mM calcium. Ritonavir inhibited cal-pain activity with increasing potency as the ritonavirconcentration was increased from 0 to 100
m
M, as shownin Fig. 2.
4. Discussion
The present study is the ®rst to indicate that ritonavir, anHIV protease inhibitor, also inhibits calcium-activatedprotease activity. This raises the possibility that ritonavirmight also exert cytoprotective effects in cells after diversetypes of insults associated with increased intracellularCa
2
where calpain activation has been shown to occurand cell survival could be increased by treatment withcalpain inhibitors. For example, calpain inhibitors havealready been shown to protect auditory sensory cells fromhypoxia and neurotrophin withdrawal-induced apoptosis[22]. Other studies have exposed cytoprotective propertiesof calpain inhibitors in models of tissue damage caused byischemic stroke [23].Concentrations of ritonavir used in this study are readilyachievable with oral dosing regimens in humans. Com-pounds such as PD150606, a calpain inhibitor, shown todecrease hypoxic/hypoglycemic and excitotoxin-mediatedneuronal injury
in vitro
, have
i
constants of 0.21 to0.37
m
M [24]. While the
i
of ritonavir is an order of magnitude higher, plasma concentrations of ritonavir usedfor anti-retroviral therapy are well within range of the
i
.Human subjects administered ritonavir orally at doses of 600 mgtwiceperdayachievedpeakplasmaconcentrationsranging from 18.6 to 46
m
M and trough concentrationsfrom 10.4 to 17.5
m
M [25].The cytoprotective effects of ritonavir in this modelsystem should prompt the investigation of its
in vivo
therapeutic ef®cacy in animal models. Many
in vivo
animal models of cerebral ischemia, shock trauma, and
Table 1Comparison of estimated
i
valuesExperiments
i
(
m
M)PC12 cell extract 11
Æ
7.0Purified m-calpain 9.2
Æ
1.2Purified
m
-calpain 5.9
Æ
1.4Values are means
Æ
SEM,
7
À
9.Fig. 2. Concentration±response curve of substrate hydrolysis inhibition byritonavir. The height of the bars represents the means
Æ
SEM of initialvelocities, relative fluorescence units per second (RFU/sec), underconditions of different inhibitor concentrations. Initial velocity at 100and 10
m
M ritonavir was significantly different from control (0
m
M) at
P
0
:
05,
8.
W. Wan, P.B. DePetrillo/Biochemical Pharmacology 63 (2002) 1481±1484
1483

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