Keto-1,3,4-oxadiazoles as cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.03.001

We have prepared a series of cathepsin K inhibitors bearing the keto-1,3,4-oxadiazole warhead capable of forming a hemithioketal complex with the target enzyme. By modifying binding moieties at the P₁, P₂, and prime side positions of the inhibitors, we have achieved selectivity over cathepsins B, L, and S, and have achieved sub-nanomolar potency against cathepsin K. This series thus represents a promising chemotype that could be used in diseases implicated by imbalances in cathepsin K activity such as osteoporosis.

Full text of DOI:10.1016/j.bmcl.2006.03.001

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2909–2914
Keto-1,3,4-oxadiazoles as cathepsin K inhibitors
James T. Palmer,a,* Bernard L. Hirschbein,a Harry Cheung,a John McCarter,a
James W. Janc,a Z. Walter Yua and Gregg Wesolowskib
aCelera Genomics, Inc., 180 Kimball Way, South San Francisco, CA 94080, USA
bDepartment of Bone Biology and Osteoporosis, Merck Research Laboratories, West Point, PA 19486, USA
Received 26 January 2006; revised 28 February 2006; accepted 1 March 2006
Available online 20 March 2006
Abstract—We have prepared a series of cathepsin K inhibitors bearing the keto-1,3,4-oxadiazole warhead capable of forming a
hemithioketal complex with the target enzyme. By modifying binding moieties at the P1, P2, and prime side positions of the inhib-
itors, we have achieved selectivity over cathepsins B, L, and S, and have achieved sub-nanomolar potency against cathepsin K. This
series thus represents a promising chemotype that could be used in diseases implicated by imbalances in cathepsin K activity such as
osteoporosis.
Ó 2006 Elsevier Ltd. All rights reserved.
Osteoporosis can be described as brittle bone disease,
manifested by loss of bone mass and an increased ten-
dency to fracture. The condition can be brought on by
an imbalance of the functions of osteoblasts, responsible
for bone formation, and osteoclasts, which are responsi-
ble for bone resorption.1 The major structural compo-
nents of extracellular bone are hydroxyapatite and
protein, of which the latter is 90% type I collagen. Dur-
ing bone resorption, multinucleated osteoclasts form
lacunae on the surface of the bone, into which protons
and proteases are secreted. This acidic environment is
suitable for the proteolytic degradation of the collagen
component.
was reported. By comparison, the lone example of a
keto-1,3,4-oxadiazole displayed only micromolar poten-
cy.8 While this ketoheterocycle has been described for
inhibition of serine proteases,9 there are no reports of
sub-nanomolar potencies against cysteine proteases.
We chose this oxadiazole functionality both for its ease
of synthesis via commercially available methyl esters
and also for the opportunity to explore prime side bind-
ing moieties (R0 in Scheme 1), wherein the inhibitors
span the active site histidinium-thiolate-containing cata-
lytic triad, anchored by a reversible hemithioketal bond.
We synthesized amino acid-derived 1,3,4-oxadiazole
building blocks as illustrated in Scheme 1. Conversion
of commercially available Boc-protected amino acids 1
to the aldehydes 2 (via Weinreb amides) proceeded
smoothly. These were used without further purification.
We prepared oxadiazoles 3 and subjected them to subse-
quent coupling with 2 using procedures outlined in Ref.
9. Conversion of the adducts 4 to the free amino alco-
hols 5 proceeded either through deprotection with HCl
in dioxane, or, for more easily handled salts, with anhy-
drous tosic acid.
Cathepsin K, a member of the papain superfamily of
cysteine proteases, is the major cysteine protease ex-
pressed in osteoclasts.2 Type I collagen is readily degrad-
ed by cathepsin K over a pH range of approximately
4–7, suggesting its potential as a target for osteoporosis
therapeutics.
Recent reports on the inhibition of cathepsin K have
focused on aldehydes,3 aminomethyl ketones,4 hydroxy-
methyl ketones,5 ketobenzoxazoles,6 and most recently
nitriles.7 In a recent article, the use of a-ketothiazoles
To probe the inhibitory effect of inhibitors targeting
only the active site cysteine, the P1 pocket, and the prime
side of cathepsin K, we oxidized small samples of pro-
tected amino alcohols 4 with Dess–Martin periodinane
(Scheme 2) to give the keto-1,3,4-oxadiazoles 6a–d. We
chose the norvaline (Nva) P1 residue as a hydrophobic
chain but without the branching that might favor serine
Keywords: Osteoporosis; Cathepsin; Cysteine protease; Inhibitor;
Ketoheterocycle; Reversible; Selective; Potent.
*
Corresponding author. Tel.: +1 650 866 6641; e-mail: jim.palmer@
celera.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.001

2910
J. T. Palmer et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2909–2914
O
O
O
N
a
b
NH2
3
R'
R'
O
N
H
R'
O
N
O
3
R'
OH
O
N
H
N
c, d
H
N
H
N
N
4
O
Boc
Boc
H
Boc
OH
e
R'
P1
N
N
P1
P1
2
1
OH
f or g
HX.H2N
O
5a: X = Cl
5b: X = TsO
R'
P1
N
N
Scheme 1. Reagents and condition: (a) NH2NH2ÆH2O; (b) HC(OCH3)3 (Ref. 9); (c) CH3NHOCH3ÆHCl, DCC, CH2Cl2; (d) LiAlH4, THF, 0 °C; (e)
BuLi, then MgBr2ÆO(CH2CH3)2, THF; (f) 4 M HCl/dioxane, CH2Cl2; (g) anhydrous TsOH, Et2O.
Dess-Martin
periodinane
OH
O
adverse effect on inhibitory potency (entries 6a and
6b), whereas relatively small substituents afford good
selectivity for cathepsin K.
H
N
H
N
O
N
O
N
Boc
Boc
R'
R'
N
N
CH2Cl2, RT
We next prepared a series of inhibitors incorporat-
ing a P2 residue known to be selective for cathepsin
K, namely the aminocyclohexylcarboxyl group (Ac6)
derivatized as the p-trifluoromethoxybenzamide, cho-
sen based on cathepsin K’s known preference for 4-
substituted benzamides.7a Synthesis of the elaborated
inhibitors is shown in Scheme 3. Transformation of
the commercially available Boc-Ac6OH, 7, proceed-
ed via benzyl ester formation to give 8, acidic
cleavage of the Boc group to yield 9, and coupling
with 4-trifluoromethoxybenzoic acid in the presence
of HATU to give 10. Conversion to the free acid
11 took place via hydrogenolysis. Coupling with
Nva-based amino alcohols 12a–h (synthesized as de-
scribed in Scheme 1) followed by oxidation to the
ketone gave the final products 13a–h, purified by
flash chromatography.
4a-d
Scheme 2. Preparation of keto-1,3,4-oxadiazoles.
6a-d
protease inhibition such as elastase or chymotrypsin.10
Prior work in our laboratories has indicated that the
Nva residue occupies similar geometry to the homophe-
nylalanine residue (phenethyl) but without the addition-
al molecular weight encumbrance that can hamper oral
bioavailability.11
The inhibitory potencies (Ki(app.))12 of these derivatives
are shown in Table 1, reflecting the greatest potency for
cathepsin K, with modest potencies for cathepsin S, and
little or no inhibition of cathepsins B and L. The lack of
P2 binding moieties in this series suggests that significant
potency can be achieved through fine tuning of the
prime side moiety, the most potent compound (6d) bear-
ing a thiophene residue. It does appear that electron-
donating groups on the aromatic ring may have an
This series was then tested as before against the panel of
cathepsins K, B, L, and S. Table 2 shows the results of
the enzymology.
Several patterns emerged from this study. First, the
selectivity of the inhibitors for cathepsin K largely par-
alleled our published observations in the nitrile warhead
series. Potency was consistently submicromolar, as
expected for benzamide derivatives of P2-Ac6 containing
cathepsin K inhibitors. The most telling observation,
however, was the effect of the ketooxazole substituent
in the prime side, wherein both size and electronic fac-
tors played a part in determining potency.
Table 1. Inhibition of cathepsins K, B, L, and S by Boc-Nva-1,3,4-
keto-oxadiazoles
Compound R0 =
Cat K Cat B Cat L Cat S
6a
33
17
>150
>150
>150
120
130
>150
>150
63
28
19
N
6b
Unsubstituted or electron-donating groups in the para
position of phenyloxadiazoles (13a, 13c) showed consis-
tent double digit nanomolar potency versus cathepsin K,
with several 100-fold selectivity over cathepsins B, L,
and S. While potency was similar for the meta-substitut-
ed examples, regardless of electronic push/pull (13b, 13e)
the selectivity was reduced. The most remarkable loss of
potency was observed in the p-substituted electron-with-
drawing example 13d, perhaps as a result of greater
O
6c
6d
5.6
130
12
S
0.81
Inhibition constants (Ki(app.)) are given in lM units. Assay methods
are described in Ref. 12.

J. T. Palmer et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2909–2914
2911
b
a
Boc
c
OH
Boc
OBzl
OBzl
OH
N
H
N
H
ClH.H2N
O
O
9
O
d
O
8
7
O
OBzl
e, f
N
H
N
H
O
O
O
F3CO
F3CO
11
10
OH
O
H
N
11 +
HOTs.H2N
O
N
O
N
R'
R'
H
N
O
N
N
F3CO
12a-k
13a-k
Scheme 3. Reagents: (a) benzyl alcohol, DCC, DMAP, CH2Cl2; (b) 4.0 M HCl/dioxane; (c) p-F3COC6H4CO2H, HATU, i-PrNEt2, DMF; (d) H2/Pd,
EtOH; (e) HATU, i-Pr2NEt, DMF; (f) Dess–Martin periodinane, CH2Cl2.
Table 2. Inhibition of cathepsins K, B, L, and S by 4-CF3OPh-Ac6-Nva-1,3,4-keto-oxadiazoles
Compound
R0 =
Cat K
0.029
Cat B
73
Cat L
19
Cat S
45
13a
O
13b
13c
13d
13e
13f
13g
13h
13i
0.037
0.022
0.55
2.1
57
7.5
37
10
20
O
23
11
13
CF3
CF3
0.054
1.3
1.1
1.4
10
3.3
17
10
0.035
0.0079
0.0033
0.0052
0.0017
0.43
0.098
0.093
0.27
0.22
4.6
0.3
0.16
2.9
1.0
0.8
0.47
0.32
0.76
0.35
O
S
13j
O
13k
Inhibition constants (Ki (app.)) are given in lM units.
hydration of the active ketone and therefore less electro-
philicity. The loss of potency created by the bulky sub-
stituent 13f suggests that this accommodation of
hindered groups in the prime side is limited to isopropyl
(13g) and smaller.
potency across all enzymes, particularly cathepsin B.
Nevertheless, at least 30-fold selectivity was achieved
in the case of the most potent example, 13i, whose elec-
tronics would also suggest favoring the ketone versus
hydrate.
The most potent compounds contained small ketoox-
adiazole substituents (13h and 13i) but with increased
Finally, the thiophene and furan derivatives 13j, 13k,
respectively, displayed the best balance of potency and

2912
J. T. Palmer et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2909–2914
Assay results for the modified P3 derivatives are shown
O
H
N
in Table 3. Interestingly, the simple carbamate 14 re-
tained good potency, but to increase potency to sub-
nanomolar levels, the introduction of an amine function
para to the carboxamide group served well.
R
7
Boc
+
OH
O
N
O
N
H
a
N
H
O
N
O
OH
HOTs.H2N
O
O
14, R = Boc
15, R = H (HCl salt)
b
Encouraged by the potency and selectivity of 16b, we
tested this compound in a bone resorption assay
measuring the inhibition of resorption of bovine bone
mediated by rabbit osteoclasts as a functional measure
of cellular potency.14 At the same time, we tested 16b
in a pharmacokinetic assay (cassette dosing in rats,
intravenous route) to establish its suitability for in vivo
efficacy studies. Table 4 indicates the results obtained.
While the potency of the compound in the bone resorp-
tion assay was still reasonably high, this represented a
significant shift between enzyme inhibition and func-
tional potency, quite possibly due to membrane
permeability.
N
N
12k
O
O
H
N
O
N
O
R3
N
H
O
N
c, d
15 + R3COCl
F
16a: R =
16a: R =
Me2N
Scheme 4. Reagents: (a) HATU, i-Pr2NEt, DMF; (b) 4.0 M HCl/
dioxane; (c) Et3N, THF; (d) Dess–Martin periodinane, CH2Cl2.
In addition, while the low clearance value of 18 mL/min/
kg was in line with our target, the relatively short mean
residence time (MRT, calculated as Vss/CL) indicated
that improvements would need to be made to consider
the series for evaluation in efficacy models. (Table 5).
selectivity within the series, setting the stage for subse-
quent improvements.
In an attempt to improve potency to the sub-nanomolar
level for cathepsin K inhibitors, mindful of potential
shifts between enzyme potency and potency in cellular
systems,we explored variations at the P3 position. Be-
cause of the potential for reduced functional selectivity
and/or lysosomotropism in cysteine protease inhibitors
bearing both positive charges and lipophilic moieties,13
our goal was to increase potency without resorting to
aliphatic amine groups at P3. Synthesis of this series
(Scheme 4) proceeded in a similar manner to that previ-
ously described, except that the P3 moiety was intro-
duced at the penultimate step. Coupling of 7 with 12k
to give 14, followed by HCl deprotection to yield 15,
coupling with the appropriate P3 benzoyl chloride, and
oxidation gave derivatives 16a and 16b.
To this end, we chose to lengthen the P1 residue by an
additional carbon atom, so as to ‘fine tune’ hydrophobic
interactions in the S1 pocket without adding too much
molecular weight. We prepared the norleucine series
(Nle) in a similar manner to the Nva series described
above, starting with commercially available materials.
We consistently found an increase in potency over the
shorter chain by using the four carbon substituent, sug-
gesting a subtle but significant binding effect of this res-
idue as compared to the three carbon chain. We
continued to use the furoyl group on the oxadiazole por-
tion of the inhibitor series and explored additional P2
and P3 residues to enhance potency and selectivity.
Table 3. Modifications at P3 for P3-Ac6-Nva-1,3,4-ketooxadiazoles
Compound
P3 residue
Cat K
Cat B
7.3
Cat L
18
Cat S
0.96
O
O
14
0.043
O
16a
16b
0.019
0.95
0.73
2.9
0.14
0.70
F
O
0.00095
0.96
Me2
N
Inhibition constants (Ki(app.)) are given in lM units.
Table 4. In vitro, biology, rat PK analysis of 16b
Bone res. IC50 (lM)
Cmax (lM)
CL (min/mL/kg)
18 3.2
Vss (mL/kg)
658 68
MRT (min)
37 5.2
0.132
2.86 0.55
PK conditions: formulation: 0.5 mg/mL/compound in DMSO/Cremophor EL/PEG 400/Water 15/5/20/60, pH 4.7. WinNonlin PK Analyses: 2
compartmental. 1/y weighting.

J. T. Palmer et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2909–2914
Table 5. P3-P2-Nle-1,3,4-ketooxadiazoles, inhibition of cathepsins K, B, L, and S
2913
P2
O
H
N
X
O
N
O
P3
O
N
Compound
P3
P2
X
K
B
L
S
17
None
CH2
CH2
O
52
110
150
9.2
2.6
2
18
19
20
21
22
23
None
None
None
9.6
25
34
16
48
100
28
3
O
0.5
14
0.38
0.24
H
N
F3
C
(S)-CH
(S)-CH
C
0.12
2.1
CBZ
<0.00025
<0.00025
0.002
0.001
1.3
<0.00025
0.3
O
N
H
Ac6
0.075
F3CO
Inhibition constants (Ki (app.)) are given in lM units.
2. Bromme, D.; Okamoto, K.; Wang, B. B.; Biroc, S. J. Biol.
Chem. 1996, 271, 2126.
While able to achieve sub-micromolar potency with the
simple isobutyl carbamate 20, we realized the necessity
of a designed P3 unit for sub-nanomolar potency, pref-
erably with a hydrogen bond acceptor in the form of a
carbamate or amide carbonyl; the simple trifluoroethyl-
amine P3 (21) was not beneficial in this series. The CBZ-
Leu derivative 22 proved to be relatively non-selective
against cathepsins S and L, in line with published series
cited earlier. We were gratified, however, to see that our
optimal P2 residue and P3 trifluoromethoxybenzamide
group (23) yielded an even more potent and selective
compound than we had seen in the case of the Nva series
(13k), We thus illustrated that in this series, tuning of P3,
P2, P1, and prime side binding moieties, coupled with an
activating electrophile, the 1,3,4-ketooxadiazole, yields
potent and selective cathepsin K inhibitors that may
be useful in treating osteoporosis and other diseases
involving collagen breakdown.
3. Votta, B. J.; Levy, M. A.; Badger, A.; Bradbeer, J.; Dodds,
R. A.; James, I. E.; Thompson, S.; Bossard, M. J.; Carr,
T.; Conner, J. R.; Tomaszek, T. A.; Szewczuk, L.; Drake,
F. H.; Veber, D. F.; Gowen, M. J. Bone Mineral Res.
1997, 12, 1396.
4. (a) Marquis, R. W.; Ru, Y.; LoCastro, S. M.; Zeng, J.;
Yamashita, D. S.; Oh, H.-J.; Erhard, K. F.; Davis, L. D.;
Tomaszek, T. A.; Tew, D.; Salyers, K.; Proksch, J.; Ward,
K.; Smith, B.; Levy, M.; Cummings, M. D.; Haltiwanger,
R. C.; Trescher, G.; Wang, B.; Hemling, M. E.; Quinn, C.
J.; Cheng, H-Y.; Lin, F.; Smith, W. W.; Janson, C. A.;
Zhao, B.; McQueney, M. S.; D’Alessio, K.; Lee, C.-P.;
Marzulli, A.; Dodds, R. A.; Blake, S.; Hwang, S.-H.;
James, I. E.; Gress, C. J.; Bradley, B. R.; Lark, M. W.;
Gowen, M. J. Med. Chem. 2001, 44, 1380; (b) Marquis, R.
W.; Ru, Y.; Yamashita, D. S.; Oh, H.-J.; Yen, J.;
Thompson, S. K.; Carr, T. J.; Levy, M. A.; Tomaszek,
T. A.; Ijames, C. F.; Smith, W. W.; Zhao, B.; Janson, C.
A.; Abdel-Meguid, S. S.; D’Alessio, K. J.; McQueney, M.
S.; Veber, D. F. Bioorg. Med. Chem. 1999, 7, 581; (c)
Marquis, R. W.; Yamashita, D. S.; Ru, Y.; LoCastro, S.
M.; Oh, H.-J.; Erhard, K. F.; DesJarlais, R. L.; Head, M.
S.; Smith, W. W.; Zhao, B.; Janson, C. A.; Abdel-Meguid,
S. S.; Tomaszek, T. A.; Levy, M. A.; Veber, D. F. J. Med.
Chem. 1998, 41, 3563.
References and notes
1. Peck, W. A.; Burckhardt, P.; Christiansen, C.; Fleisch, H.
A.; Genant, H. K.; Gennari, C.; Martin, T. J.; Martini, L.;
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P. H.; Young, R. T. T.; Barrett-Connor, E.; Brandi, M. L.;
Chesnut, C. H.; Delmas, P. D.; Heaney, R. P.; Johnston,
C. C.; Kanis, J. A.; Lindsay, R.; Meunier, P. J.; Papapo-
ulos, S. E.; Reginster, J. Y.; Riggs, B. L.; Riis, B. J.;
Seeman, E.; Wasnich, R. D. Am. J. Med. 1993, 94, 646.
5. Mendonca, R. V.; Venkatraman, S.; Palmer, J. T. Bioorg.
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6. McGrath, M. E.; Sprengeler, P. A.; Hill, C. M.; Martic-
honok, V.; Cheung, H.; Somoza, J. R.; Palmer, J. T.; Janc,
J. W. Biochemistry 2003, 42, 15018.

2914
J. T. Palmer et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2909–2914
7. (a) Palmer, J. T.; Bryant, C.; Wang, D-X.; Davis, D. E.;
amino-4-methylcoumarin which was monitored fluoro-
metrically (excitation at 355 nm and emission at 460 nm)
using an FMAX 96-well plate reader from Molecular
Devices (Sunnyvale, CA, USA) interfaced with a Macin-
tosh computer. Under these experimental conditions 1 lM
of product produces a fluorescence signal of approximate-
ly 125 U. The velocity of the enzyme-catalyzed reaction
was obtained from the linear portion of the progress
curves (usually the first 5 min after addition of substrate).
Apparent inhibition constants, Ki(app.)’s, were calculated
from the velocity data generated at the various inhibitor
concentrations using the software package, BatchKi
(obtained from Dr. Petr Kuzmic, Biokin Ltd., Pullman,
WA, USA). BatchKi provides a parametric method for the
determination of inhibitor potency using a transformation
of the tight binding inhibition model described by Mor-
rison15 and further refines the appKi values by nonlinear
least-squares regression. The apparent inhibition constant
is related to the true thermodynamic binding constant, Ki,
by the following relationship: Ki = appKi/(1 + [substrate]/
Km) (2). Since substrates are typically supplied in the
assays at their Km, Ki = appKi/2. Specific assay conditions
for each cathepsin tested are as follows: cathepsin K: The
buffer used to assay this enzyme consisted of: 50 mM MES
(pH 5.5), 2.5 mM D,L-dithiothreitol (DTT), 2.5 mM eth-
ylenediaminetetraacetic acid (EDTA), and 10% dimethyl-
Setti, E. L.; Rydzewski, R. M.; Venkatraman, S.;
Tian, Z-Q.; Burrill, L. C.; Mendonca, R. V.; Springman,
E.; McCarter, J.; Chung, T.; Cheung, H.; McGrath, M.;
Somoza, J.; Enriquez, P.; Yu, Z. W.; Strickley, R. M.; Liu,
L.; Venuti, M. C.; Percival, M. D.; Falgueyret, J-P.; Prasit,
P.; Oballa, R.; Riendeau, D.; Young, R. N.; Wesolowski,
G.; Rodan, S. B.; Johnson, C.; Kimmel, D. B.; Rodan, G.
J. Med. Chem. 2005, 48, 7520; (b) Black, W. C.; Bayly, C.
I.; Davis, D. E.; Demarais, S.; Falgueyret, J-P.; Leger, S.;
Li, C. S.; Masse, F.; McKay, D. J.; Palmer, J. T.; Percival,
M. D.; Robichaud, J.; Tsou, N.; Zamboni, R. Bioorg.
Med. Chem. Lett. 2005, 15, 4741; (c) Robichaud, J.;
Oballa, R.; Prasit, P.; Falgueyret, J-P.; Percival, M. D.;
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Bryant, C.; Venkatraman, S.; Setti, E.; Mendonca, R.;
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8. Tavares, F. X.; Deaton, D. N.; Miller, A. B.; Miller, L. R.
Bioorg. Med. Chem. Lett. 2005, 15, 3891.
9. Ohmoto, K.; Yamamoto, T.; Okuma, M.; Horiuchi, T.;
Imanishi, H.; Odagaki, Y.; Kawabata, K.; Sekioka, T.;
Hirota, Y.; Matsuoka, S.; Nakai, H.; Toda, M.; Cheronis,
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Chem. 2001, 44, 1268.
10. See, for example: (a) Steitz, T. A.; Shulman, R. G. Annu.
Rev. Biophys. Bioeng. 1982, 11, 419; (b) Vajda, T.; Szabo,
T. Acta Biochim. Biophys. Acad. 1976, 11, 287.
sulfoxide (DMSO). Recombinant cathepsin
K was
11. Lipinski, C. A. Adv. Drug Del. Rev. 1997, 23, 3.
12. Enzyme assays. All enzymes used in these studies, with the
exception of human cathepsin B, were produced by Celera
Genomics. Cathepsin B was from human liver and was
purchased from Athens Research and Technology (Ath-
ens, GA, USA). The substrates used in these studies were
purchased from the following vendors: Z-Phe-Arg-AMC,
Boc-Leu-Lys-Arg-AMC, and Z-Val-Val-Arg-AMC were
from Bachem (Torrance, CA, USA) and Z-Leu-Arg-AMC
was from Calbiochem-Novabiochem (San Diego, CA,
USA). Bovine serum albumin was purchased from the
Sigma Chemical Company (St. Louis, MO, USA). Rou-
tine buffer components and all other chemicals used in
these experiments were of the highest available quality.
Enzyme inhibition studies were performed under several
sets of conditions; each set was tailored to provide the
optimal activity of the given cathepsin being assayed. Each
cathepsin was incubated with the inhibitor, present at
variable concentrations, under the conditions specified
below. Enzyme and inhibitor were incubated together for
30 min at room temperature (21–24 °C) in 96-well
U-bottomed, microtiter plates (Falcon, from Becton–
Dickinson, Franklin Lakes, NJ, USA). After the pre-
incubation phase, reactions were initiated with the addi-
tion of the 7-amino-4-methylcoumarin (AMC) substrate
specified below. The hydrolysis of these substrates yields 7-
supplied at 1 nM. Substrate, Z-Phe-Arg-AMC, was sup-
plied at 40 lM. Human cathepsin B: the buffer used to
assay this enzyme consisted of: 50 mM MES (pH 6.0),
2.5 mM DTT, 2.5 mM EDTA, 0.001 Tween 20, and 10%
DMSO. Human liver cathepsin B was supplied at 1 nM.
Substrate, Boc-Leu-Lys-Arg-AMC, was supplied at
190 lM. Human cathepsin L: the buffer used to assay this
enzyme consisted of: 50 mM MES (pH 5.5), 2.5 mM DTT,
2.5 mM EDTA, and 10% DMSO. Recombinant human
cathepsin L was supplied at 500 pM. Substrate, Z-Phe-
Arg-AMC, was supplied at 10 lM. Human cathepsin S:
the buffer used to assay this enzyme consisted of: 50 mM
MES (pH 6.5), 2.5 mM b-mercaptoethanol (BME),
2.5 mM EDTA, 100 mM NaCl, 0.001% bovine serum
albumin (BSA), and 10% DMSO. Recombinant human
cathepsin S was supplied at 500 pM. Substrate, Z-Val-Val-
Arg-AMC, was supplied at 60 lM.
13. Falgueyret, J.-P.; Desmarais, S.; Oballa, R.; Black, W. C.;
Cromlish, W.; Khougaz, K.; Lamontagne, S.; Masse, F.;
Riendeau, D.; Toulmond, S.; Percival, M. D. J. Med.
Chem. 2005, 48, 7535.
14. Falgueyret, J. P.; Oballa, R. M.; Okamoto, O.; Wesolow-
ski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.;
Riendeau, D.; Rodan, S. B.; Percival, M. D. J. Med.
Chem. 2001, 44, 94.
15. Morrison, J. F. Biochim. Biophys. Acta 1969, 185, 269.