Inhibition of cathepsin D by tripeptides containing statine analogs

Article abstract of DOI:10.1016/S0006-2952(99)00103-3

Various analogs of statine, a remarkable amino acid component of the protease inhibitor pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and HIV-1 protease. Copyright (C) 1999 Elsevier Science Inc.

Full text of DOI:10.1016/S0006-2952(99)00103-3

Biochemical Pharmacology, Vol. 58, pp. 329–333, 1999.
© 1999 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/99/$–see front matter
PII S0006-2952(99)00103-3
SHORT COMMUNICATION
Inhibition of Cathepsin D by Tripeptides Containing
Statine Analogs
Michel Bessodes,*† Kostas Antonakis,* Jean Herscovici,* Marcel Garcia,‡
࿣
࿣
Henri Rochefort,‡ Franc¸oise Capony,§ Yves Lelie`vre and Daniel Scherman
*CNRS UMR 133, ENSCP, 75005 PARIS; ‡INSERM U148, 34090 MONTPELLIER; §INSERM U454, HOPITAL
࿣
ˆ
ARNAUD DE VILLENEUVE, 34295 MONTPELLIER; AND RHONE POULENC-RORER, E-94403 VITRY, FRANCE
ABSTRACT. Various analogs of statine, a remarkable amino acid component of the protease inhibitor
pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and
HIV-1 protease. BIOCHEM PHARMACOL 58;2:329–333, 1999. © 1999 Elsevier Science Inc.
KEY WORDS. statine; inhibitor; cathepsin D; tripeptide; protease; cell proliferation
Inoperable invasive slow-growing tumors are a major chal-
lenge in cancer therapy. The proteases produced by cancer
cells and involved in extracellular matrix digestion play a
well-established and important role in the tumor invasion
mechanism [1, 2]. Matrix protease inhibitors can therefore
be considered as potential antimetastatic and antitumor
therapeutic agents [3]. Thus, some inhibitors of collagenase
type metalloproteases are currently under clinical investi-
gation and have yielded promising results [4]. Collagenases
are involved in extracellular fiber degradation, but other
proteases could well be involved in the degradation of
attachment proteins that link the cells to the matrix.
It has been shown, for instance, that the level of the
aspartyl protease cathepsin D is consistently more impor-
tant in some breast invasive tumors than in normal tissues.
This overexpression has been correlated to the risk of
metastasis in several clinical studies [5–7]. The deregulated
overexpression of cathepsin D by transfection in rat tumor
cells increases their metastatic potential via a mechanism
probably requiring cathepsin D catalytic activity [8]. More-
over, cathepsin D is mitogenic in different cell types, and
different substrates which could be maturated by the pro-
tease, such as growth inhibitors or precursors of growth
factors, are proposed to mediate this activity [9]. Some
studies also pointed to the mitogenic activity of the
secreted procathepsin D that can be internalized in cancer
cells via different membrane receptors [10–12]. This sug-
gests the implication of this protease in the metastatic
progression of invasive tumors.
nanomolar range [14, 15]. Thus, pepstatine (Fig. 1) or its
analogs could be good candidates for antiproliferative
activity. One of the major problems, however, remains the
poor solubility and membrane permeation of pepstatine, as
well as its lack of selectivity among aspartyl proteases,
which precludes its use as a therapeutic agent. A tremen-
dous amount of work has been done on the design and
synthesis of new analogs, with the general aim of improving
the bioavailability and selectivity of these compounds. For
our part, we have described a new route to statine [16, 17],
one of the most interesting amino acid components of
pepstatine, which allows the synthesis of a wide variety of
analogs. We present in this paper the biological results
obtained with a representative family of tripeptides con-
taining statine analogs.
MATERIALS AND METHODS
Synthesis of Statine Analogs and their Incorporation
into Tripeptides
The synthetic methods and characterizations described
briefly here have been detailed elsewhere [17]. We prepared
a series of statine analogs (Fig. 2) with various C-terminal
substituents and side chains and with different configura-
tions, starting from isovaleraldehyde or hexanal. The cor-
responding allylic alcohols obtained via a Grignard reaction
were then enantioselectively epoxidized [18, 19]. As it has
been described that the hydroxyl function is essential for
the activity [20], a blank compound, deprived of this
hydroxyl, was prepared by hydrogenation of the allylic
alcohol and amination of the resulting saturated alcohol.
The epoxy–alcohols were subsequently transformed into
the epoxy–azides with inverted configurations [21]. The
compounds without the C-carboxyl group were obtained by
lithium aluminum hydride (LAH) reduction of the epoxy–
azides to the corresponding hydroxy–amines. The cyano
compounds were obtained by nucleophilic addition of
Pepstatine is a natural pentapeptide isolated from various
species of Actinomyces [13]. It is a good inhibitor of most
aspartyl proteases, including cathepsin D, with a Ki in the
† Corresponding author: Dr. M. Bessodes, UMR133, ENSCP 11 rue Pierre
et Marie Curie, 75005 Paris, France. Tel. 33 (1) 53 10 12 95; FAX 33 (1)
53 10 12 93; E-mail: bessodes@infobiogen.fr
Received 25 September 1998; accepted 26 January 1999.

330
M. Bessodes et al.
300 nm. The effectiveness of the compounds on cell
proliferation was determined on a metastatic and high
cathepsin D-expressing cell line MDA-MB-231 after 6 days
in 5% fetal calf serum by measuring DNA synthesis as
previously described [9]. Detection of the effects of the
cathepsin D inhibitors was made possible by using the
lowest serum concentration supporting maximal prolifera-
tion.
FIG. 1. Structure of pepstatine.
cyanide anion to the corresponding epoxides. The resulting
nitriles were then hydrolyzed to the acids and esterified
with 2-(trimethylsilyl)ethanol and trimethylchlorosilane.
The structures and enantiomeric purity of the compounds
were determined by high resolution NMR.
It has been shown previously that tripeptides correspond-
ing to the initial sequence of pepstatine, although less
active than the original pentapeptide, could advanta-
geously be used for the preliminary evaluation of new
statine derivatives [22]. Therefore, we condensed three
different dipeptides with the synthesized amines to give the
corresponding tripeptides using PyBOP (benzotriazol-1-
yloxy tripyrrolidinophosphonium hexafluoro phosphate) as
the activator in a well-established route. The products were
then purified by reversed phase HPLC (Fig. 3).
RESULTS AND DISCUSSION
The analogs of statine were condensed with the C-terminal
side of three different N-carbobenzyloxydipeptides: N-
CBZ-Val-Val (8, 9, 10, 15, 18, 21); N-CBZ-Val-Phe (11,
12, 16, 19); and N-CBZ-Val-Trp (13, 14, 17, 20). The
resulting tripeptides therefore included three distinctive
types of derivatives:
—A blank compound (8), lacking the hydroxyl function
on the statine analog;
—Analogs with a linear alkyl side chain, a deoxy termina-
tion, and of different stereochemistry 9, 11, 13, (S, S) and
10, 12, 14 (R, R);
—Analogs bearing the isopropyl side chain of statine with
different functions on the terminal side: 15, 16, 17 (deoxy
termination); 18, 19, 20 (cyano terminal group); and 21
(ester group).
Enzymatic and Biochemical Procedures
Cathepsin D inhibition was evaluated in two ways: 1) The
radioactivity of the acid-soluble supernatant of the diges-
tion products of [14C] methemoglobin was measured in the
presence of different concentrations of inhibitors [23]; and
2) The difference in optical density observed at 300 nm,
after cleavage of the Glu-Phe(NO2) bond of the 0.1 mM
substrate Pro-Thr-Glu-Phe(NO2)-Arg-Leu by a 47 nM
cathepsin D, compared to a blank experiment, was corre-
lated to the percent of inhibition at different concentra-
tions of inhibitors. The same assay method was used with a
100 nM HIV-1 protease which cleaves between Phe(NO2)
and Pro, the 0.4 mM substrate Val-Ser-Gln-Asn-
Phe(NO2)-Pro-Ile-Val, with an optical density increase at
The first results of inhibition on cathepsin D were obtained
for compounds 8, 9, 15, and 21 (Fig. 4). [14C] Methemo-
globin was used as a substrate with different concentrations
of inhibitors. As expected, compound 8 was not active and
thus could be used as a blank in the following experiments.
Three different analogs of statine incorporated into CBZ-
Val-Val dipeptides 9, 15, and 21 gave the same activity.
Then, the antiproliferative properties of compounds 8, 9,
15, and 18 were tested on the growth of MDA/MB231
cancer cells. Again compound 8 was inactive, while com-
pounds 9 and 18 showed significant activity (Fig. 5).
Compound 15 appeared inactive whereas its inhibitory
FIG. 2. (i) N3H, DEAD, Ph3P; (ii)
Ti(IV)OiPr4, D- or L-diisopropyl tar-
trate, tBHP; (iii) H2, Pd/C; (iv) Li-
AlH4, diethyl ether; (v) KCN, EtOH;
(vi) Na2O, tBuOH, H2O; (vii) 2-(tri-
methylsilyl) ethanol, trimethylchlorosi-
lane.

Inhibition of Cathepsin D
331
FIG. 3. Structure of the different peptides
prepared for the evaluation of the synthe-
sized statine derivatives.
activity on methemoglobin was similar to that of com-
pounds 9 and 18. This suggests differences in the bioavail-
ability of these compounds. The fact that pepstatine was
inactive at these concentrations (data not shown) also
confirmed its well-known difficulty in penetrating into
intact cells.
A systematic evaluation of the inhibitory potencies of all
the compounds synthesized was made on cathepsin D and
FIG. 4. Inhibition of cathepsin D-induced proteolysis of methe-
moglobin in vitro by statine derivatives.
FIG. 5. Antiproliferative activities of statine derivatives on
MDA-MB-231 breast cancer cells.

332
M. Bessodes et al.
TABLE 1. Inhibition (%) of cathepsin D and HIV protease I
tions, this could suggest that cellular uptake of these
compounds remains a limiting factor in their action, and
thus improvement in their membrane permeation should be
considered. On the other hand, since tripeptides are ac-
tively absorbed through the transepithelial barrier of the
gastrointestinal track, these new products could be prom-
ising as orally absorbed inhibitors of extracellular cathepsin
D and as such for the therapy of some invasive tumors and
metastases or for inflammation treatment.
by the tripeptides containing statine derivatives
Cathepsin D
inhibition
(%)
HIV protease
inhibition
(%)
Compound (mM)
V-V analogs
8 10Ϫ5
20
ND
91
25
99
31
99
32
99
28
9
24
22
11
8
10Ϫ7
9 10Ϫ5
(S-S) 10Ϫ7
15 10Ϫ5
(S-S) 10Ϫ7
18 10Ϫ5
This work was supported by the CNRS and Rhoˆne-Poulenc Rorer. We
thank the Association pour la Recherche sur le Cancer (ARC) for
financial support. We are grateful to D. Derocq for his excellent
assistance in proliferation studies.
(S-S) 10Ϫ7
21 10Ϫ5
(S-S) 10Ϫ7
V-F analogs
11 10Ϫ5
93
13
12
8
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