Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cellpermeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cellpermeant epoxysuccinyl based cathepsin B inhibitor [RGlyGlyLeu(2S, 3S)tEpsLeuProOH; R=OMe] with the C-terminal heptapeptide segment of penetratin (R=εAhxArg ArgNleLysTrpLysLysNH(2)). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodruglike epoxysuccinyl peptides E64d and CA074Me

Assfalg-Machleidt, Irmgard

Deluca, Dominga

Höhneke, Clara

Machleidt, Werner

Schaschke, Norbert

Sommerhoff, Christian P.

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

iologically contributing to lysosomal protein degradation,this protease has been shown to be membrane-associat-ed and/or secreted into the pericellular space in severaltumor cell lines and is believed to promote metastasisand tumor progression (Elliott and Sloane, 1996; Froschet al., 1999). Moreover, according to very recent findingscytosolic cathepsin B seems to play a role in apoptoticcell death (Guicciardi et al., 2000; Foghsgaard et al.,2001; Mathiasen et al., 2001).In various attempts to elucidate the contribution ofcathepsin B to these processes, specific inhibitors andaffinity labels have been used. Depending on the locali-sation of cathepsin B in the pericellular/extracellularspace or within the cell, it is of great importance to mod-ulate the membrane permeability of these tools. In orderto target extracellular cathepsin B, we have recently in-troduced the endo-epoxysuccinyl peptide 1 as well asaffinity labels (compounds 2 and 3) derived from thisirreversible inhibitor (Figure 1) (Schaschke et al., 1998,2000a). These highly cathepsin B-selective and potentinhibitors are based on the CA030-like fragment HO-(2S,3S)-tEps-Leu-Pro-OH as lead structure, which waselongated by the peptide stretch Leu-Gly-Gly derivedfrom the cathepsin B propeptide (amino acids 46 – 48) tosimultaneously address interactions with both the S andthe S‘ subsites. A special feature of this parent inhibitor isthat the terminal glycine residue is located solvent-ex-posed at the surface of the enzyme as proposed by mod-eling experiments (data not shown). Consequently, thisposition offers the possibility to further derivatise the par-ent inhibitor 1 with various functional groups without af-fecting its inhibitory potency. This concept has been con-firmed by synthesis of the affinity labels 2 and 3 as well asof a cathepsin B-selective drug carrier system consistingof β-cyclodextrin as carrier- and the inhibitor as address-unit (Schaschke et al., 2000b).To render inhibitor 1 membrane-permeable two gener-al strategies seem promising. On one hand it seems pos-sible to convert 1 into a permeable prodrug-like form bysynthesising the proline methyl ester. This approach hasbeen successfully applied to transform the well-knowncathepsin B inhibitor CA074 into the cell permeable pro-inhibitor CA074Me (Buttle et al., 1992). On the otherhand, an alternative strategy is to conjugate the inhibitorwith a cell-penetrating peptide (Lindgren et al., 2000).Here, we report the synthesis and functional characteri-sation of a penetratin-epoxysuccinyl peptide conjugateBiol. Chem., Vol. 383, pp.849 – 852, May 2002 · Copyright © by Walter de Gruyter · Berlin · New YorkNorbert Schaschke1, Dominga Deluca1, IrmgardAssfalg-Machleidt2, Clara Höhneke3, ChristianP. Sommerhoff3 and Werner Machleidt2,*1 Max-Planck-Institut für Biochemie, Am Klopferspitz 18a,D-82152 Martinsried, Germany2 Adolf-Butenandt-Institut für Physiologische Chemie,Physikalische Biochemie und Zellbiologie der Ludwig-Maximilians-Universität, Schillerstrasse 42, D-80336 München, Germany3 Abteilung für Klinische Chemie und KlinischeBiochemie, Chirurgische Klinik Innenstadt, Klinikum derLudwig-Maximilians-Universität, Nußbaumstr. 20, D-80336 München, Germany* Corresponding authorBesides its physiological role in lysosomal proteinbreakdown, extralysosomal cathepsin B has recentlybeen implicated in apoptotic cell death. Highly specificirreversible cathepsin B inhibitors that are readily cell-permeant should be useful tools to elucidate the ef-fects of cathepsin B in the cytosol. We have covalentlyfunctionalised the poorly cell-permeant epoxysucci-nyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminalheptapeptide segment of penetratin (R=Ahx-Arg-Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory po-tency and selectivity for cathepsin B versus cathepsinL of the parent compound was not affected by the con-jugation with the penetratin heptapeptide. The conju-gate was shown to efficiently penetrate into MCF-7cells as an active inhibitor, thereby circumventing anintracellular activation step that is required by other in-hibitors, such as the prodrug-like epoxysuccinyl pep-tides E64d and CA074Me.Key words: Antennapedia /CA074 /Cathepsin L /MCF-7 cells.Papain-like cysteine proteases are involved in diversephysiological processes and, if disregulated, they con-tribute to a variety of disorders (Kirschke et al., 1995; Ottoet al., 1997; Barrett et al., 1998; Brömme, 1999). In par-ticular, cathepsin B is proteolytically active in differentcompartments, depending on its role under physiologicalor pathological conditions (Mort and Buttle, 1997). Phys-Short CommunicationEpoxysuccinyl Peptide-Derived Cathepsin B Inhibitors:Modulating Membrane Permeability by Conjugation withthe C-Terminal Heptapeptide Segment of PenetratinBereitgestellt von | Universitaetsbibliothek der LMU MuenchenAngemeldet | 129.187.254.47Heruntergeladen am | 18.11.13 13:43derived from inhibitor 1 following the concept of effectorfunctionalisation outlined above.The 16-mer peptide (amino acids 43 – 58, RQIKIWFQ-NRRMKWKK) derived from the third helix of the home-odomain of the Antennapedia protein, also called pene-tratin, is widely used for the intracellular delivery of pep-850 N. Schaschke et al.Fig. 1 Structure of Epoxysuccinyl Peptide 1 and the Effector-Functionalised Inhibitors 2, 3 and 4.Fig. 2 Solid-Phase Synthesis of the Epoxysuccinyl Peptide/Penetratin HP Conjugate 4.Reaction conditions: step (i), a. piperidine/NMP (1:5), b. Fmoc-AA/HBTU/HOBt/DIEA (4:4:4:8), NMP; 8 cycles of double coupling; step(ii), a. piperidine/NMP (1:5), b. HO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OtBu/HBTU/HOBt/DIEA (1.5:1.5:3), NMP; step (iii), 95% aq.TFA/TIS (98.5:1.5). The synthesis was performed using a rink amide MBHA resin.Bereitgestellt von | Universitaetsbibliothek der LMU MuenchenAngemeldet | 129.187.254.47Heruntergeladen am | 18.11.13 13:43tides and oligonucleotides (Derossi et al., 1998). Recent-ly, by an extensive SAR study it was shown that the C-ter-minal heptapeptide segment of penetratin (amino acids52 – 58, RRMKWKK, penetratin HP) is sufficient for effi-cient cell membrane translocation (Fischer et al., 2000).Furthermore, it was shown that Met-54 is freely ex-changeable with either Leu or Nle (Fischer et al., 2000).The truncated penetratin, in which Met-54 is exchangedagainst Nle, is very attractive because the synthetical ef-fort is drastically reduced and the resulting peptide is ox-idation-insensitive. Therefore we have chosen this partic-ular new vector for our study.The penetratin heptapeptide was synthesised by stan-dard Fmoc/tBu chemistry on solid support (Figure 2). Af-ter introducing ε-amino hexanoic acid as an additionalspacer, the suitably protected inhibitor [HO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OtBu] obtained by solution syn-thesis as described previously (Schaschke et al., 2000a)was coupled as fragment using HBTU/HOBt. The finalcleavage/deprotection was performed with 95% aq TFAcontaining 1.5% triisopropylsilane yielding conjugate 4,as characterised by RP-HPLC, ESI-MS and amino acidanalysis.The second-order rate constants of inactivation ofcathepsin B and L by conjugate 4 as well as by inhibitor 1and the affinity labels 2 and 3 are summarised in Table 1.Comparison of the rate constants for cathepsin B inacti-vation by the parent inhibitor 1 and the labels 2 and 3clearly reveals that the functionalisation with reportergroups has no effect on the inhibitory potency. In thecase of the new conjugate 4 the inhibitory potency iseven slightly increased upon covalent attachment of thepenetratin segment, presumably due to additional inter-actions of the highly positively charged penetratin hep-tapeptide portion with the protein surface. The heptapep-tide itself has no inhibitory activity against cathepsin B (Ki≥240 µM; data not shown). The selectivity for cathepsin Bversus cathepsin L is not affected by the conjugation. Thedata obtained with the new conjugate 4 support our de-sign concept and characterise the parent inhibitor 1 as astructure privileged for modifications due to its particularbinding mode.Membrane permeability studies were performed withMCF-7 breast cancer cells as described previously(Schaschke et al., 2000a). The results are summarised inFigure 3. The covalent functionalisation of the parent in-hibitor with the penetratin segment leads to an efficientcell membrane translocation of the cargo molecule andfully confirms the reported data on the potential of thepenetratin heptapeptide (Fischer et al., 2000). Whereasincubation (30 min) of MCF-7 cells with 1 µM inhibitor 1does not effect inactivation of intracellular cathepsin B, aEpoxysuccinyl Peptide-Penetratin Conjugates 851Table 1 Second-Order Rate Constants for the Inactivation of Cysteine Proteases by the Penetratin Pentapeptide (Penetratin HP)-Functionalised Epoxysuccinyl Peptide.Inhibitor Cathepsin B Cathepsin L Ratio CB/CLk2/Ki [M– 1s– 1] k2/Ki [M– 1s– 1]MeO-Gly-Gly-Leu(2S,3S)-tEps-Leu-Pro-OH (1) 1520000±88 800 1204±29 1262Rhodamine B-NH-(CH2)6-NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH (2) 1530000±83 500 323±30 4736Biotin-NH-(CH2)6-NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH (3) 1726000±40900 256±14 6742Penetratin HP-CO-(CH2)5-NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH (4) 6100000±270000 3590±154 1699The k2/Ki-value for compound 4 was determined in 250 mM sodium acetate buffer pH 5.5 as described previously (Schaschke et al.,1998) and is the mean ± SD from 7 experiments after correction for substrate competition. The inhibitory potencies of compounds 1, 2and 3 (Schaschke et al., 1998, 2000a) are listed for comparison.Fig. 3 Cell Permeability of Epoxsuccinyl-Peptides.Cell permeability was determined with MCF-7 breast cancercells essentially as described previously (Schaschke et al.,2000a). Confluent cells grown in 24-well plates were incubatedfor 30 min at 37°C with 300 µl of serum-free medium containing0.5% DMSO without or with increasing concentrations of in-hibitor 1 (0.1, 1.0, 10, and 50 µM) or penetratin-functionalised in-hibitor 4 (0.01, 0.1, 0.3, 1.0 and 10 µM). Thereafter, the cells werewashed five times with PBS and lysed in 200 µl of 50 mM sodiumacetate, pH 5.5, 0.5% Triton X-100, 2 mM EDTA for 30 min atroom temperature. Residual cathepsin B activity in the lysateswas measured with the substrate Z-Arg-Arg-NHMec (50 µM) fol-lowed by inhibition with compound 1. The columns representmean values of three experiments with standard deviations(bars). Incubation of the cells with the penetratin heptapeptide it-self (at concentrations ≤10 µM) had no effect on residual cathep-sin B activity (data not shown).Bereitgestellt von | Universitaetsbibliothek der LMU MuenchenAngemeldet | 129.187.254.47Heruntergeladen am | 18.11.13 13:43concentration of 0.3 µM of conjugate 4 results in almostcomplete inactivation. A concentration as low as 0.01 µMis sufficient to block approx. 60% of the intracellularcathepsin B activity. As expected, the heptapeptide itselfhas no effect on intracellular cathepsin B activity (datanot shown). In contrast to prodrug-like epoxysuccinylpeptides, such as E64d and CA074Me, conjugate 4 per-meates the membrane as an active inhibitor, thus circum-venting an intracellular activation step by deesterifica-tion.In conclusion, the data obtained from inhibition kinet-ics and cell permeability experiments classify the newepoxysuccinyl peptide-penetratin conjugate 4 as a po-tent tool for inactivation of intracellular cathepsin B. Incombination with inhibitor 1 and the affinity labels 2 and3 this set of compounds provides a promising tool kit toselectively investigate the roles of intracellular and peri-cellular cathepsin B.AcknowledgementsThe authors wish to thank Prof. L. Moroder for generous sup-port. This work was supported by the SFB 469 of the Ludwig-Maximilians-Universität München, Germany (grants A2, A6 andB6).ReferencesBarrett, A.J., Rawlings, N.D., and Woessner Jr., J.F. (1998).Handbook of Proteolytic Enzymes (London, UK: AcademicPress).Buttle D.J., Murata, M., Knigth, C.G., and Barrett, A.J. (1992).CA074 Methyl ester: a proinhibitor for intracellular cathepsinB. Arch. Biochem. Biophys. 299, 377 – 380.Brömme, D. (1999). Cysteine proteases as therapeutic targets.Drug News Perspect. 12, 73 – 82.Derossi, D., Chassaing, G., and Prochiantz, A. (1998). Trojanpeptides: the penetratin system for intracellular delivery.Trends Cell Biol. 8, 84 – 87.Elliott, E., and Sloane, B.F. (1996). The cysteine protease cathep-sin B in cancer. Perspect. Drug Discov. Design 6, 12 – 32.Fischer, P.M., Zhelev, N.Z., Wang, S., Melville, J.E., Fåhraeus, R.,and Lane, D.P. (2000). Structure-activity relationship of trun-cated and substituted analogues of the intracellular deliveryvector penetratin. J. Peptide Res. 55, 163 – 172.Foghsgaard, L., Wissing, D., Mauch, D., Lademann, U.,Bastholm, L., Boes, M., Elling, F., Leist, M., and Jäättelä, M.(2001). Cathepsin B acts as a dominant execution protease intumor cell apoptosis induced by tumor necrosis factor. J. CellBiol. 153, 999 – 1009.Frosch, B.A., Berquin, I., Emmert-Buck, M.R., Moin, K., andSloane, B.F. (1999). Molecular regulation, membrane associa-tion and secretion of tumor cathepsin B. APMIS 107, 28 – 37.Guicciardi, M.E., Deussing, J., Miyoshi, H., Bronk, S.F., Svingen,P.A., Peters, C., Kaufmann, S.H., and Gores, G.J. (2000).Cathepsin B contributes to TNF-α-mediated hepatocyteapoptosis by promoting mitochondrial release of cytochromec. J. Clin. Invest. 106, 1127 – 1137.Kirschke, H., Barrett, A.J., and Rawlings, N.D. (1995). Lysosomalcysteine proteinases. In: Protein Profile, Vol. 2, P. Sheterline,ed. (London, UK: Academic Press), pp. 1587 – 1643.Lindgren, M., Hällbrink, M., Prochiantz, A., and Langel, Ü.(2000). Cell-penetrating peptides. Trends Pharmacol. Sci. 21,99 – 103.Mathiasen, I.S., Hansen, C.M., Foghsgaard, L., and Jäättelä, M.(2001). Sensitization to TNF-induced apoptosis by 1,25-dihy-droxy vitamin D3 involves up-regulation of the TNF receptor 1and cathepsin B. Int. J. Cancer 93, 224 – 231.Mort, J.S., and Buttle, D.J. (1997). Molecules in focus. Cathep-sin B. Int. J. Biochem. Cell Biol. 29, 715 – 720.Otto, H.-H., and Schirmeister, T. (1997). Cysteine proteases andtheir inhibitors. Chem. Rev. 97, 133 – 171.Schaschke, N., Assfalg-Machleidt, I., Machleidt, W., and Mo-roder, L. (1998). Substrate/propeptide-derived endo-epoxy-succinyl peptides as highly potent and selective cathepsin Binhibitors. FEBS Lett. 421, 80 – 82.Schaschke, N., Assfalg-Machleidt, I., Laßleben, Th., Sommer-hoff, C.P., Moroder, L., and Machleidt, W. (2000a). Epoxysuc-cinyl peptide-derived affinity labels for cathepsin B. FEBSLett. 482, 91 – 96.Schaschke, N., Assfalg-Machleidt, I., Machleidt, W., Laßleben,Th., Sommerhoff, C.P., and Moroder, L. (2000b). β-Cyclodex-trin/epoxysuccinyl peptide conjugates: a new drug targetingsystem for tumor cells. Bioorg. Med. Chem. Lett. 10, 677 – 680.Received October 18, 2001; accepted November 14, 2001852 N. Schaschke et al.Bereitgestellt von | Universitaetsbibliothek der LMU MuenchenAngemeldet | 129.187.254.47Heruntergeladen am | 18.11.13 13:43

accepted November 14,

CA074 Methyl ester: a proinhibitor for intracellular cathepsin B.

Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor.

Cell-penetrating peptides.

contributes to TNF-α -mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c.

Cysteine proteases and their inhibitors.

Cysteine proteases as therapeutic targets.

Epoxysuccinyl peptide-derived affinity labels for cathepsin B.

Lysosomal cysteine proteinases. In:

Molecular regulation, membrane association and secretion of tumor cathepsin B.

Molecules in focus.

Sensitization to TNF-induced apoptosis by 1,25-dihydroxy vitamin

Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector penetratin.

Substrate/propeptide-derived endo-epoxysuccinyl peptides as highly potent and selective cathepsin B inhibitors.

Trojan peptides: the penetratin system for intracellular delivery.

β -Cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells.

Epoxysuccinyl peptide-derived cathepsin B inhibitors: Modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin

Schaschke N, Deluca D, Assfalg-Machleidt I, Hohneke C, Sommerhoff CP, Machleidt W. Epoxysuccinyl peptide-derived cathepsin B inhibitors: Modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin. BIOLOGICAL CHEMISTRY. 2002;383(5):849-852

Deluca, D

Assfalg-Machleidt, I

Hohneke, C

Sommerhoff, CP

Machleidt, W

Publications at Bielefeld University

Universität München: Elektronischen Publikationen

iologically contributing to lysosomal protein degradation,
this protease has been shown to be membrane-associat-
ed and/or secreted into the pericellular space in several
tumor cell lines and is believed to promote metastasis
and tumor progression (Elliott and Sloane, 1996; Frosch
et al., 1999). Moreover, according to very recent findings
cytosolic cathepsin B seems to play a role in apoptotic
cell death (Guicciardi et al., 2000; Foghsgaard et al.,
2001; Mathiasen et al., 2001).
In various attempts to elucidate the contribution of
cathepsin B to these processes, specific inhibitors and
affinity labels have been used. Depending on the locali-
sation of cathepsin B in the pericellular/extracellular
space or within the cell, it is of great importance to mod-
ulate the membrane permeability of these tools. In order
to target extracellular cathepsin B, we have recently in-
troduced the endo-epoxysuccinyl peptide 1 as well as
affinity labels (compounds 2 and 3) derived from this
irreversible inhibitor (Figure 1) (Schaschke et al., 1998,
2000a). These highly cathepsin B-selective and potent
inhibitors are based on the CA030-like fragment HO-
(2S,3S)-tEps-Leu-Pro-OH as lead structure, which was
elongated by the peptide stretch Leu-Gly-Gly derived
from the cathepsin B propeptide (amino acids 46 – 48) to
simultaneously address interactions with both the S and
the S‘ subsites. A special feature of this parent inhibitor is
that the terminal glycine residue is located solvent-ex-
posed at the surface of the enzyme as proposed by mod-
eling experiments (data not shown). Consequently, this
position offers the possibility to further derivatise the par-
ent inhibitor 1 with various functional groups without af-
fecting its inhibitory potency. This concept has been con-
firmed by synthesis of the affinity labels 2 and 3 as well as
of a cathepsin B-selective drug carrier system consisting
of β-cyclodextrin as carrier- and the inhibitor as address-
unit (Schaschke et al., 2000b).
To render inhibitor 1 membrane-permeable two gener-
al strategies seem promising. On one hand it seems pos-
sible to convert 1 into a permeable prodrug-like form by
synthesising the proline methyl ester. This approach has
been successfully applied to transform the well-known
cathepsin B inhibitor CA074 into the cell permeable pro-
inhibitor CA074Me (Buttle et al., 1992). On the other
hand, an alternative strategy is to conjugate the inhibitor
with a cell-penetrating peptide (Lindgren et al., 2000).
Here, we report the synthesis and functional characteri-
sation of a penetratin-epoxysuccinyl peptide conjugate
Biol. Chem., Vol. 383, pp.849 – 852, May 2002 · Copyright © by Walter de Gruyter · Berlin · New York
Norbert Schaschke1, Dominga Deluca1, Irmgard
Assfalg-Machleidt2, Clara Höhneke3, Christian
P. Sommerhoff3 and Werner Machleidt2,*
1 Max-Planck-Institut für Biochemie, Am Klopferspitz 18a,
D-82152 Martinsried, Germany
2 Adolf-Butenandt-Institut für Physiologische Chemie,
Physikalische Biochemie und Zellbiologie der 
Ludwig-Maximilians-Universität, Schillerstrasse 42, 
D-80336 München, Germany
3 Abteilung für Klinische Chemie und Klinische
Biochemie, Chirurgische Klinik Innenstadt, Klinikum der
Ludwig-Maximilians-Universität, Nußbaumstr. 20, 
D-80336 München, Germany
* Corresponding author
Besides its physiological role in lysosomal protein
breakdown, extralysosomal cathepsin B has recently
been implicated in apoptotic cell death. Highly specific
irreversible cathepsin B inhibitors that are readily cell-
permeant should be useful tools to elucidate the ef-
fects of cathepsin B in the cytosol. We have covalently
functionalised the poorly cell-permeant epoxysucci-
nyl-based cathepsin B inhibitor [R-Gly-Gly-Leu-(2S,
3S)-tEps-Leu-Pro-OH; R=OMe] with the C-terminal
heptapeptide segment of penetratin (R=Ahx-Arg-
Arg-Nle-Lys-Trp-Lys-Lys-NH2). The high inhibitory po-
tency and selectivity for cathepsin B versus cathepsin
L of the parent compound was not affected by the con-
jugation with the penetratin heptapeptide. The conju-
gate was shown to efficiently penetrate into MCF-7
cells as an active inhibitor, thereby circumventing an
intracellular activation step that is required by other in-
hibitors, such as the prodrug-like epoxysuccinyl pep-
tides E64d and CA074Me.
Key words: Antennapedia /CA074 /Cathepsin L /
MCF-7 cells.
Papain-like cysteine proteases are involved in diverse
physiological processes and, if disregulated, they con-
tribute to a variety of disorders (Kirschke et al., 1995; Otto
et al., 1997; Barrett et al., 1998; Brömme, 1999). In par-
ticular, cathepsin B is proteolytically active in different
compartments, depending on its role under physiological
or pathological conditions (Mort and Buttle, 1997). Phys-
Short Communication
Epoxysuccinyl Peptide-Derived Cathepsin B Inhibitors:
Modulating Membrane Permeability by Conjugation with
the C-Terminal Heptapeptide Segment of Penetratin
Bereitgestellt von | Universitaetsbibliothek der LMU Muenchen
Angemeldet | 129.187.254.47
Heruntergeladen am | 18.11.13 13:43
derived from inhibitor 1 following the concept of effector
functionalisation outlined above.
The 16-mer peptide (amino acids 43 – 58, RQIKIWFQ-
NRRMKWKK) derived from the third helix of the home-
odomain of the Antennapedia protein, also called pene-
tratin, is widely used for the intracellular delivery of pep-
850 N. Schaschke et al.
Fig. 1 Structure of Epoxysuccinyl Peptide 1 and the Effector-Functionalised Inhibitors 2, 3 and 4.
Fig. 2 Solid-Phase Synthesis of the Epoxysuccinyl Peptide/Penetratin HP Conjugate 4.
Reaction conditions: step (i), a. piperidine/NMP (1:5), b. Fmoc-AA/HBTU/HOBt/DIEA (4:4:4:8), NMP; 8 cycles of double coupling; step
(ii), a. piperidine/NMP (1:5), b. HO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OtBu/HBTU/HOBt/DIEA (1.5:1.5:3), NMP; step (iii), 95% aq.
TFA/TIS (98.5:1.5). The synthesis was performed using a rink amide MBHA resin.
Bereitgestellt von | Universitaetsbibliothek der LMU Muenchen
Angemeldet | 129.187.254.47
Heruntergeladen am | 18.11.13 13:43
tides and oligonucleotides (Derossi et al., 1998). Recent-
ly, by an extensive SAR study it was shown that the C-ter-
minal heptapeptide segment of penetratin (amino acids
52 – 58, RRMKWKK, penetratin HP) is sufficient for effi-
cient cell membrane translocation (Fischer et al., 2000).
Furthermore, it was shown that Met-54 is freely ex-
changeable with either Leu or Nle (Fischer et al., 2000).
The truncated penetratin, in which Met-54 is exchanged
against Nle, is very attractive because the synthetical ef-
fort is drastically reduced and the resulting peptide is ox-
idation-insensitive. Therefore we have chosen this partic-
ular new vector for our study.
The penetratin heptapeptide was synthesised by stan-
dard Fmoc/tBu chemistry on solid support (Figure 2). Af-
ter introducing ε-amino hexanoic acid as an additional
spacer, the suitably protected inhibitor [HO-Gly-Gly-Leu-
(2S,3S)-tEps-Leu-Pro-OtBu] obtained by solution syn-
thesis as described previously (Schaschke et al., 2000a)
was coupled as fragment using HBTU/HOBt. The final
cleavage/deprotection was performed with 95% aq TFA
containing 1.5% triisopropylsilane yielding conjugate 4,
as characterised by RP-HPLC, ESI-MS and amino acid
analysis.
The second-order rate constants of inactivation of
cathepsin B and L by conjugate 4 as well as by inhibitor 1
and the affinity labels 2 and 3 are summarised in Table 1.
Comparison of the rate constants for cathepsin B inacti-
vation by the parent inhibitor 1 and the labels 2 and 3
clearly reveals that the functionalisation with reporter
groups has no effect on the inhibitory potency. In the
case of the new conjugate 4 the inhibitory potency is
even slightly increased upon covalent attachment of the
penetratin segment, presumably due to additional inter-
actions of the highly positively charged penetratin hep-
tapeptide portion with the protein surface. The heptapep-
tide itself has no inhibitory activity against cathepsin B (Ki
≥240 µM; data not shown). The selectivity for cathepsin B
versus cathepsin L is not affected by the conjugation. The
data obtained with the new conjugate 4 support our de-
sign concept and characterise the parent inhibitor 1 as a
structure privileged for modifications due to its particular
binding mode.
Membrane permeability studies were performed with
MCF-7 breast cancer cells as described previously
(Schaschke et al., 2000a). The results are summarised in
Figure 3. The covalent functionalisation of the parent in-
hibitor with the penetratin segment leads to an efficient
cell membrane translocation of the cargo molecule and
fully confirms the reported data on the potential of the
penetratin heptapeptide (Fischer et al., 2000). Whereas
incubation (30 min) of MCF-7 cells with 1 µM inhibitor 1
does not effect inactivation of intracellular cathepsin B, a
Epoxysuccinyl Peptide-Penetratin Conjugates 851
Table 1 Second-Order Rate Constants for the Inactivation of Cysteine Proteases by the Penetratin Pentapeptide (Penetratin HP)-
Functionalised Epoxysuccinyl Peptide.
Inhibitor Cathepsin B Cathepsin L Ratio CB/CL
k2/Ki [M– 1s– 1] k2/Ki [M– 1s– 1]
MeO-Gly-Gly-Leu(2S,3S)-tEps-Leu-Pro-OH (1) 1520000±88 800 1204±29 1262
Rhodamine B-NH-(CH2)6-NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH (2) 1530000±83 500 323±30 4736
Biotin-NH-(CH2)6-NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH (3) 1726000±40900 256±14 6742
Penetratin HP-CO-(CH2)5-NH-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH (4) 6100000±270000 3590±154 1699
The k2/Ki-value for compound 4 was determined in 250 mM sodium acetate buffer pH 5.5 as described previously (Schaschke et al.,
1998) and is the mean ± SD from 7 experiments after correction for substrate competition. The inhibitory potencies of compounds 1, 2
and 3 (Schaschke et al., 1998, 2000a) are listed for comparison.
Fig. 3 Cell Permeability of Epoxsuccinyl-Peptides.
Cell permeability was determined with MCF-7 breast cancer
cells essentially as described previously (Schaschke et al.,
2000a). Confluent cells grown in 24-well plates were incubated
for 30 min at 37°C with 300 µl of serum-free medium containing
0.5% DMSO without or with increasing concentrations of in-
hibitor 1 (0.1, 1.0, 10, and 50 µM) or penetratin-functionalised in-
hibitor 4 (0.01, 0.1, 0.3, 1.0 and 10 µM). Thereafter, the cells were
washed five times with PBS and lysed in 200 µl of 50 mM sodium
acetate, pH 5.5, 0.5% Triton X-100, 2 mM EDTA for 30 min at
room temperature. Residual cathepsin B activity in the lysates
was measured with the substrate Z-Arg-Arg-NHMec (50 µM) fol-
lowed by inhibition with compound 1. The columns represent
mean values of three experiments with standard deviations
(bars). Incubation of the cells with the penetratin heptapeptide it-
self (at concentrations ≤10 µM) had no effect on residual cathep-
sin B activity (data not shown).
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concentration of 0.3 µM of conjugate 4 results in almost
complete inactivation. A concentration as low as 0.01 µM
is sufficient to block approx. 60% of the intracellular
cathepsin B activity. As expected, the heptapeptide itself
has no effect on intracellular cathepsin B activity (data
not shown). In contrast to prodrug-like epoxysuccinyl
peptides, such as E64d and CA074Me, conjugate 4 per-
meates the membrane as an active inhibitor, thus circum-
venting an intracellular activation step by deesterifica-
tion.
In conclusion, the data obtained from inhibition kinet-
ics and cell permeability experiments classify the new
epoxysuccinyl peptide-penetratin conjugate 4 as a po-
tent tool for inactivation of intracellular cathepsin B. In
combination with inhibitor 1 and the affinity labels 2 and
3 this set of compounds provides a promising tool kit to
selectively investigate the roles of intracellular and peri-
cellular cathepsin B.
Acknowledgements
The authors wish to thank Prof. L. Moroder for generous sup-
port. This work was supported by the SFB 469 of the Ludwig-
Maximilians-Universität München, Germany (grants A2, A6 and
B6).
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852 N. Schaschke et al.
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Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cellpermeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cellpermeant epoxysuccinyl based cathepsin B inhibitor [RGlyGlyLeu(2S, 3S)tEpsLeuProOH; R=OMe] with the Cterminal heptapeptide segment of penetratin (R=[epsilon]AhxArg ArgNleLysTrpLysLysNH(2)). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodruglike epoxysuccinyl peptides E64d and CA074Me

Schaschke, N.

Deluca, D.

Assfalg-Machleidt, I.

Höhneke, C.

Sommerhoff, C. P.

Machleidt, W.

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Epoxysuccinyl peptide-derived cathepsin B inhibitors: Modulating membrane permeability by conjugation with the C- terminal heptapeptide segment of penetratin

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Epoxysuccinyl peptide-derived cathepsin B inhibitors: Modulating membrane permeability by conjugation with the C-terminal heptapeptide segment of penetratin

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