Interaction of Papain-Like Cysteine Proteases
with Dipeptide-Derived Nitriles
Reik Löser,1 Klaus Schilling,2 and Michael Gütschow1
1Pharmaceutical Institute, Poppelsdorf, University of Bonn, D-53115 Bonn, Germany
2Institute of Biochemistry I, Klinikum, Friedrich-Schiller-University,
Nonnenplan 2, D-07743 Jena, Germany
Thiol-dependent cathepsins represent a class of mammalian cysteine proteases which are mainly located in lysosomes.
These enzymes are involved in several cellular functions and play a major role in pathological processes. Cathepsin K is
abundantly expressed in human osteoclasts and possesses a collagenolytic activity. Inhibitors against cathepsin K are
drug candidates for osteoporosis. Cathepsin L is suggested to be involved in rheumatoid arthritis as well as in tumor
invasion and metastasis. Cathepsin S is crucial for MHC class II-mediated antigen presentation. Among other
cathepsins, these three proteases are target enzymes in the development of inhibitors [1]. Amino acid- and dipeptidederived nitriles are known to inhibit cathepsins as well as the related plant cysteine protease papain. For this interaction,
the reversible formation of a covalent thioimidate intermediate was demonstrated by NMR, binding kinetics and X-ray
crystallographic studies [2]. Using the mixed acid anhydride method, different dipeptide nitriles with Gly in P1 and
varying amino acids in P2 position were synthesized and evaluated for their potency to inhibit papain, cathepsin L,
cathepsin S, and cathepsin K, respectively. The kinetic results obtained with papain were compared to those with the
mammalian proteases in order to prove the utility of papain as a model enzyme [3] for the development of peptidederived nitrile inhibitors. This investigation was performed to support our efforts to design irreversible nitrile-based
inhibitors starting from potent reversible inhibitors of cysteine proteases [4].
1. O
O
2.
R
R
THF
O
O
N
H
rt
-25 °C
H
N
1
3. aq. H2NCH2CN * 0.5 H2SO4 / NaOH
OH
N
H
R2
O
R
1
O
O
Cl
2
O
N
N
O
Scheme 1 Synthesis of dipeptide nitriles
Tab. 1 Inhibition of papain, cathepsin L, cathepsin S and cathepsin K
inhibition of
R1
compd.
papain
bovine
cathepsin L
human
cathepsin S
human
cathepsin K
Ki´ (LM)
Ki (LM)
Ki (LM)
Ki (LM)
R2
1
t-Bu
i-Pr
2.4 ± 0.1
2.8 ± 0.1
6.2 ± 0.4
6.5 ± 0.4
2
t-Bu
i-Bu
2.4 ± 0.1
1.2 ± 0.1
0.13 ± 0.01
0.12 ± 0.01
3
Bn
i-Bu
3.7 ± 0.2
0.75 ± 0.02
0.13 ± 0.01
0.035 ± 0.001
4
t-Bu
sec-Bu
32 ± 11
4.9 ± 0.5
4.1 ± 0.1
5.8 ± 0.3
5
t-Bu
CH2-cyclopropyl
16 ± 11
47 ± 10
0.65 ± 0.04
3.3 ± 0.2
6
t-Bu
CH2-cyclohexyl
2.6 ± 0.1
1.1 ± 0.1
0.044 ± 0.001
5.3 ± 0.2
7
t-Bu
(CH2)2SMe
0.79 ± 0.01
19 ± 11
0.66 ± 0.03
0.73 ± 0.03
H
N
N
8
O
> 100
> 100
35 ± 11
t-Bu
Bn
0.32 ± 0.01
0.52 ± 0.02
0.62 ± 0.05
4.08 ± 0.21
10
Bn
Bn
0.43 ± 0.01
0.18 ± 0.02
0.51 ± 0.05
0.26 ± 0.01
11
t-Bu
CH2(p-OH)Ph
0.23 ± 0.01
0.40 ± 0.02
0.97 ± 0.09
30 ± 21
12
t-Bu
CH2-indol-3´-yl
9.2 ± 0.2
0.35 ± 0.01
0.24 ± 0.02
29 ± 61
13
t-Bu
CH2-thionaphthen-3´-yl
9.0 ± 0.2
0.32 ± 0.02
0.30 ± 0.01
34 ± 31
14
t-Bu
CH2-2´-thienyl
0.23 ± 0.01
1.6 ± 0.1
0.27 ± 0.01
1.2 ± 0.1
15
t-Bu
CH2-3´-thienyl
0.69 ± 0.01
4.5 ± 0.4
0.76 ± 0.03
2.1 ± 0.1
16
t-Bu
CH2-2´-furyl
1.5 ± 0.1
14 ± 11
2.8 ± 0.2
4.1 ± 0.2
17
t-Bu
CH2(p-F)Ph
0.33 ± 0.01
0.64 ± 0.07
0.59 ± 0.03
13 ± 11
18
t-Bu
CH2(m-F)Ph
0.61 ± 0.01
0.70 ± 0.04
0.86 ± 0.06
6.3 ± 0.3
19
t-Bu
CH2(o-F)Ph
0.49 ± 0.02
0.47 ± 0.03
0.77 ± 0.07
3.3 ± 0.3
20
t-Bu
CH2C6F5
> 10
2.5 ± 0.3
15 ± 11
> 100
O
O
10
N
H
> 100
N
N
> 100
> 100
8
15
10
6
15
pK i
5 7
5
9
21
5
6
> 100
10
5
7
N
O
O
8
10
8
5
4
15
15
20
20
3
1
2
3
4
5
6
7
co
m
8
human
cathepsin K
human
cathepsin S
20
9
po
un
d
10
11
12
13
20
bovine
cathepsin L
14
15
16
17
18
papain
19
20
21
> 100
O
Fig. 1 Inhibition of papain, cathepsin L, cathepsin S, and cathepsin K by dipeptide nitriles 1-21
A series of 21 dipeptide nitriles were synthesized by reacting urethane-protected L-amino acids with glycine nitrile in the
presence of N-methylmorpholine and isobutyl chloroformate to obtain 1-21 in 12-91 % yield. These compounds were
evaluated towards their potency to inhibit papain, bovine cathepsin L, human cathepsin S and human cathepsin K. In
spite of the covalent nature of the enzyme-inhibitor interaction, the kinetic data correspond to competitive inhibition.
This is in accordance to the reversibility of the modification of the active site thiol.
HS-Cys
P2
N
H
H
N
O
P2
N
N
H
P1
S
H
N
O
Cys
NH
It is known that papain-like proteases are characterized by a primary specificity determined by S2-P2 site interactions.
Aliphatic, aromatic and heteroaromatic residues were selected as substituents for the P2-position. In addition, a fluorine
scan for Phe in P2 was performed to evaluate the fluorophilic potential of the S2-pocket. Strong inhibition of papain was
achieved with dipeptide nitriles bearing an aromatic substituent for R2. Cathepsin L followed this trend, but accepted like cathepsin S - also bulky condensed heteroaromatic residues like the indol moiety of 12 and the thionaphthene
moiety of 13, while these residues were not favoured by cathepsin K. Cathepsin S prefered aliphatic and - slightly less
efficient - aromatic residues in P2. The most potent and selective inhibitor for this enzyme was obtained by placing a
cyclohexyl moiety into that position, as realized in compound 6. Replacement of the thienyl residue in 14 by furyl in 16
lead to a significant loss of potency towards cathepsin S. Cathepsin K favoured aliphatic residues in the P2 position and
its best inhibitor of this series was compound 3. Cathepsin K also had a remarkable preference for aromatic residues in
P3, which became obvious by comparing the leucine derivatives 2 and 3.
P1
Scheme 2 Interaction of cysteine proteases with dipeptide nitriles via reversible thioimidate formation
B)
3
[12]=0
[12]=0.3 LM
500
2
2.5
[12]=0.7 LM
[12]=1.5 LM
[12]=2.0 LM
400
1.5
[12]=3.0 LM
[12]=5.0 LM
300
100
0
100
time (s)
150
200
1.5
1
0
1
0.5
50
The overlapping P2-specificity of the cathepsins L, S and K complicates the development of selective inhibitors.
However, selectivity was achieved by introducing non-proteinogenic amino acids into P2, as it could be observed for 5, 6
and 14. The proline derivative 8 was selective for cathepsin K, but showed low affinity. The fluorine scan did not reveal
any surprise. With respect to the nonfluorinated compound 9, monofluorination was well tolerated in all cases, while
multiple fluorination leads to a remarkable loss of affinity (compound 20). N-Methylation of the amide bond between P1
and P2 resulted in a dramatic decrease in affinity (compound 21).
2
0.5
200
0
1/rate (s)
[12]=1.0 LM
rate (1/s)
intensity of fluorescence emission (fu)
A)
0
0
1
2
1
3
2
3
4
[14] (LM)
4
5
5
6
6
[14] (LM)
Fig. 2 A) Monitoring of the bovine cathepsin L-catalyzed hydrolysis of Z-Phe-Arg-NHMec (10 LM) in the presence of
increasing concentrations of compound 12 (100 mM sodium phosphate, pH 6.0, 100 mM NaCl, 5 mM EDTA, 25 LM DTT,
1 % DMSO, 0.01 % brij, 37 °C). The reaction was initiated by addition of the enzyme, which was activated with DTT in a
preincubation over 1 min. Data were obtained by measuring the fluorescence emission at 440 nm after excitation at 360 nm.
The other cathepsins were measured in similar manner using Z-Val-Val-Arg-NHMec and Z-Leu-Arg-NHMec as substrates
for cathepsin S and cathepsin K, respectively. For papain a spectrophotometric assay was choosen, using Z-Phe-Arg-NHNp
as substrate.
B) Plot of the rates of hydrolysis of Z-Phe-Arg-NHMec versus concentrations of compound 12. The linear dependence
shown in the Dixon plot (insert) indicates competitive inhibition. Non-linear regression gave an apparent inhibition constant
Ki’ = (1 + [S]/Km) Ki = 1.3 ± 0.1 LM. A Michaelis constant Km = 3.8 ± 0.1 LM was determined separately.
Acknowledgement
The authors are grateful to Prof. Dr. B. Wiederanders, Jena, and Prof. Dr. D. Brömme, Mount Sinai School of
Medicine, New York, for providing cathepsin S and cathepsin K, respectively. Mrs. E. Dimmig, Jena, is
acknowledged for skillful assistance in the fluorimetric enzyme assays.
References
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