WO2003042239A1 - Peptides inhibiting signaling by ras-like gtpases - Google Patents

Abstract

A peptide comprising an amino acid sequence corresponding to a protein transduction domain (PTD) which has cell-penetrating capacity and a amino acid sequence corresponding to a variable part of the C-terminus of a Ras-like GTPase which has Ras-like GTPase signaling inhibiting capacity. Said peptide for use in a method to inhibit cellular functions mediated by the Ras-like GTPase. Pharmaceutical compositions comprising said peptide. A method for manufacturing an inhibitor of a Ras-like GTPase, comprising analyzing said Ras-like GTPase to determine the amino acid sequence located between the final alpha helix, counting from the N- to the C-terminus, and the CAAX-box or simular motif, if present, or the C-terminus itself when no CAAX-box or simular motif is present, selecting an amino acid sequence corresponding to a natural or artificial protein transduction domain (PTD), and producing a peptide comprising said selected amino acid sequence corresponding to a PTD, which has cell-penetrating capacity, said determined amino acid sequence corresponding to a variable part of the C-terminus of said Ras-like GTPase, which has capacity to inhibit signaling by said Ras-like GTPase, and optionally a spacer between both amino acid sequences.

Description

Title: Peptides inhibiting signaling by Ras-like GTPases

FIELD OF THE INVENTION

The present invention concerns synthetic peptides having a sequence which represents a 'protein transduction' domain (PTD) linked to a part of the C-terminus of a human small GTP-binding protein. The PTD may for example be derived from the Human Immunodeficiency Virus-Tat protein, the Antennapedia homeodomain protein or the Vsp22 protein from Herpes Simplex Virus or represent an artificial sequence with protein transduction capacity. The small GTP binding proteins are members of the superfamily of Ras-like small GTPases that show significant homology throughout most of their sequence to the prototype GTPase, p21Ras. It has been established that the hypervariable C-termini of these GTPases can act as a selective inhibitor. This invention therefore represents a collection of cell-penetrating inhibitors of Ras-like GTPases. These inhibitors are ideal tools to study intracellular signaling, they may be helpful in defining the actions of new drugs and may represent new drugs themselves.

BACKGROUND OF THE INVENTION

Protein Transduction

The efficiency of introduction of small therapeutic drugs into cells relies on their hydrophilic and hydrophobic properties, or on those of a (chemically) added carrier, for instance a lipid. The notion that certain small peptides can also function as such a carrier was derived from studies on the Human Immunodeficiency Virus (HIV) Tat protein (Green and Loewenstein, 1988;

Frankel and Pabo, 1988) which is capable to spontaneously enter mammalian cells. This transfer phenomenon was mapped to a small region of 11 amino acids, designated the 'protein transduction domain' (PTD; see US 5,652,122 and WO99/29721). The covalent linkage of other peptides or proteins to this PTD, either by molecular biological or chemical methods, confers the cell- penetrating capacity to the fusion product (Nagahara et al., 1998).

In addition to the HIV-Tat peptide, other PTD's have been defined. These are derived from either naturally occurring proteins, eg. the Antennapedia homeodomain or the Vsp22 protein from Herpes Simplex Virus (HSV; Ford et al., 2001; Vocero-Akbani et al., 2001). Furthermore, the realization that the protein transduction capacity of these sequences is dependent on their alpha helical nature, has stimulated research directed at defining additional, artificial, alpha-helical peptides that show improved protein transduction properties (Ho et al., 2001).

The PTD enters cells without being dependent on any known carrier or receptor system, which makes the technique of protein transduction applicable to any cell type. The entrance of fusion peptides or fusion proteins into cells occurs within a matter of minutes. Moreover, protein transduction provides approximately 100% efficiency, which represents a major improvement over classical methods such as microinjection, or molecular technologies such as transfection of DNA constructs or retroviral transduction. Finally, protein transduction, although based on naturally occurring proteins (or parts thereof) does not involve the introduction of possibly dangerous mutations in the genome of the host cell. The technique has been shown to be applicable to a wide range of proteins and to be effective in vitro as well as in vivo (Schwarze et al, 1999; Ford et al., 2001; Vocero-Akbani et al., 2001).

Ras like- small GTP binding proteins

There are at least two large families of GTP-binding proteins that are important for cellular function. One of these is the famliy of α-subunits (approximately 40 kD) of the heterotrimeric G-proteins, that can associate with and mediate signaling by seven-times-membrane-spanning receptors. The other concerns the superfamily of Ras-like GTP-binding and -hydrolysing proteins (Ras-like GTPases). These are small (approximately 21 kD) GTPases that shuttle between the cytosol and the plasma membrane, where they activate signaling pathways through selective interactions with effector molecules. According to generally accepted models, the binding to GTP instead of to GDP, catalyzed by so-called GTP-exchange proteins, is accompanied by or subsequent to translocation to the plasma membrane (this translocation is reported for some of the Ras like proteins, for instance the Rho-related GTPases) and induces conformational changes that allow activation of downstream signaling events. The Ras superfamily is subdivided into a series of families named after their first or best-described member: Ras, Rho, Rab, Ran, Sar/Arf (Takai et al., 2001). In addition to these, a variety of Ras-like (sometimes coined 'hypothetical') proteins have been identified in the human genome of which no functional information is currently available.

Within the above mentioned families, as well as between some of these, the protein members show amino acid sequence homologies, spanning most of the proteins, that can be very high. As a result of this, the overall topology of the Ras-like GTPases is very similar. The region of highest diversity in primary sequence is found in the hypervariable C-termini. There is little knowledge on the role of this region in GTPase functioning, and most relevant information on this point concerns the role of the C-terminus in membrane targeting. Given the low homology of this region, we hypothesize that the C-terminus may play a role in the specificity of signaling by Ras-like GTPases.

During synthesis, the Ras-like GTPases undergo various types of modifi- cation at the C-terminus. The extreme C-terminal amino acids (for most of the GTPases defined as the CAAX box, where C is cysteine; A is aliphatic, and X is any amino acid) are truncated by proteolysis up to the cysteine, which becomes carboxymethylated and to which a lipid anchor is attached through the action of specific lipid transferases. There are various types of lipid involved and the C-termini of the various GTPases therefore carry different lipid modifications such as palmitoyl, farnesyl or geranyl-geranyl, depending on their amino acid sequences. These lipid anchors, together with additional lipid moieties that can be linked to additional cysteines, located further upstream in some GTPases, are important for proper membrane localization. This membrane localization is essential for normal function because the targets of the small GTPases reside mainly at the (plasma) membrane (Takai et al., 2001). For instance, one can reduce tumor cell growth by inhibitors of farnesyl transferase, which mediates the farnesylation of the Ras C-terminus (Sebti and Hamilton, 2000). The specificity of these agents and the redundancy of the cellular machinery, (i.e. other lipids can be added instead of the farnesyl, resulting in a more or less normal functioning GTPase) are the major drawbacks of this approach (Sebti and Hamilton, 2000).

In addition to the lipid moieties, the polybasic region, which occurs in the C-termini of many of the Ras-like GTPases, has also been implicated in the membrane association of these proteins, due to the positive charge which allows interaction with the acidic phospholipid environment of the plasma membrane. Recent studies on a limited set of Ras isoforms suggests that the variability of the C-terminal region and its associated variation in lipid modification may direct Ras proteins to specific microdomains in the plasma membrane or to other cellular membranes, such as of mitochondia, thereby allowing the proteins to interact with a subset of target proteins. In this way, intracellular targeting may also be important for the specificity of signaling (Wolfman, 2001).

Because of the large number of known GTPases, there is probably no physiological process which does not rely on the proper functioning of one or more of these proteins. Such processes include cell growth and division, differentiation, apoptosis and migration. The importance of small GTPases is further underscored by the embryonic lethality in mice that is associated with the inactivation of the genes encoding, for instance, Ras or Racl, and the implication in the development of human cancers of a wide range of these GTPases, again with Ras as the best studied example (Bos, 1997).

GTP-binding proteins act as molecular switches: they are on in the GTP- bound state and o /in the GDP-bound state. Under resting conditions, these GTPases are kept in an inactive state by GDP-dissociation inhibitory proteins. Upon cellular stimulation, socalled exchange factors can bind and partially unfold the nucleotide-binding site, allowing dissociation of the GDP and subsequent binding of GTP, which is in approximately 10-fold excess in the cytoplasm. Inactivation, in turn, requires the intrinsic GTPase activity, which can be promoted by GTPase-activating proteins (GAP's). In the past decades, extensive research on the structure -function relationship of the small GTPases has defined a series of conserved amino acids and regions in the proteins that are crucial for specific properties, such as GTP-binding and signaling towards downstream effector proteins (Fig. 1).

As mentioned above, the different members of the small GTPase families show very high levels of sequence homology, sometimes over 50%. However, the part of the molecules which shows the highest diversity resides in the C- terminus. The current invention is based on this diversity, and makes use of the here described finding that this region can act as a selective inhibitor which, when fused to a PTD, can be used to block GTPase signaling in living cells. To our knowledge, the use of such a peptide inhibitor of GTPase signaling in live cells has not been described. SUMMARY OF THE INVENTION

This invention provides in a first aspect a peptide comprising an amino acid sequence corresponding to a protein transduction domain (PTD) which has cell-penetrating capacity and an amino acid sequence corresponding to a variable part of the C-terminus of a Ras-like GTPase which has Ras-like GTPase signaling inhibiting capacity.

In a further aspect, the invention provides such a peptide for use in a method to inhibit cellular functions mediated by the said Ras-like GTPase. The invention furthermore provides in a further aspect a method for manufacturing an inhibitor of a Ras-like GTPase, comprising analyzing said Ras-like GTPase to determine the amino acid sequence located between the final alpha helix, counting from the N- to the C-terminus, and the CAAX-box or similar motif, if present, or the C-terminus itself when no CAAX-box or similar motif is present, selecting an amino acid sequence corresponding to a natural or artificial protein transduction domain (PTD), and producing a peptide as claimed herein comprising said selected amino acid sequence corresponding to a PTD, which has cell-penetrating capacity, said determined amino acid sequence corresponding to a variable part of the C-terminus of said Ras-like GTPase, which has capacity to inhibit signaling by said Ras-like GTPase, and optionally a spacer between both amino acid sequences.

The invention provides also a method for inhibiting cellular functions mediated by a Ras-like GTPase in eukaryotic cells, preferably mammalian cells, comprising contacting said cells with a peptide as claimed herein. Furthermore, the invention provides a pharmaceutical composition comprising a peptide as claimed herein together with a pharmaceutically acceptable carrier or diluent.

The invention further relates to a method of treatment of a mammal, in particular a human being, in need of a treatment that inhibits cellular functions mediated by a Ras-like GTPase, comprising administering to said mammal a peptide as claimed herein in an effective dose to inhibit the cellular functions of said Ras-like GTPase. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1

A schematic representation of the Ras GTPase, used as a prototype for the large family of Ras-like GTPases. Essential amino acids and regions are indicated in the figure.

Figure 2

Effect of pre-incubation with 200 μg/ml Tat-Racl C-terminal peptide on Transwell migration of HL60 cells in the absence (open bars) or presence

(hatched bars) of 100 ng/ml Stromal Derived Factor 1 (SDF-1). Two hundred μg/ml Tat-Racl C-terminal peptide completely blocked the SDF-1 induced migration of HL60 cells.

Figure 3

Effect of pre-incubation with increasing concentrations of Tat-Racl C- terminal peptide on Transwell migration of HL60 cells in the absence (open bars) or presence (hatched bars) of 100 ng/ml Stromal Derived Factor 1 (SDF- 1). The Tat-PTD (negative control) did not prevent SDF-1 induced migration, while 200 μg/ml Tat-Racl C-terminal peptide efficiently blocked the SDF-1 induced migration of HL60 cells. Furthermore, there was a clear concentration-dependent inhibitory effect of the Tat-Racl C-terminal peptide on the migratory capacity of HL60 cells.

Figure 4

Effect of Tat-RhoA C-terminal peptide on the Transwell migration of HL60 cells in the absence (open bars) or presence (hatched bars) of 100 ng/ml Stromal Derived Factor 1 (SDF-1). The Tat-PTD (negative control) did not affect the spontaneous or the SDF-1 induced migration. There was a clear concentration-dependent inhibitory effect of the Tat-RhoA C-terminal peptide on the migratory behavior of HL60 cells after pre-incubation (pi). When the Tat-RhoA C-terminal peptide was present during the complete procedure (tot), it completely blocked the SDF-1 induced migration in all concentrations tested. Figure 5

Effect of increasing concentrations of the Tat-Racl C-terminal peptide and Tat-Rac2 C-terminal peptide on the spontaneous migration of KGlA cells across fibronectin-coated filters. The peptides were present during the complete procedure. Two hundred μg/ml Tat-Racl C-terminal peptide abrogated the migration of KGlA cells. Although there was a concentration- dependent effect of the Tat-Racl C-terminal peptide on the migration, substantial variation between individual samples was observed. The Tat- Rac2 C-terminal peptide appeared not to influence the migration of KGlA cells in the concentrations tested. Each condition was analysed in duplicate.

Figure 6

Effect of pre-incubation with the Tat-PTD, Tat-Racl C-terminal peptide, Tat-Rac2 C-terminal peptide and Tat-RhoA C-terminal peptide on the Transwell migration of human umbilical cord blood cells in the absence (open bars) or presence (hatched bars) of 100 ng/ml Stromal Derived Factor 1 (SDF- 1). The Tat-PTD peptide (negative control) did not affect the spontaneous migration and only slightly reduced the SDF-1 induced migration, while the Tat-Racl C-terminal peptide reduced the spontaneous migration as well as the SDF-1 induced migration. Tat-Rac2 C-terminal peptide increased the spontaneous migration but blocked the SDF-1 induced migration to the level of spontaneous migration in the presence of Tat-Rac2 C-terminal peptide. Tat-RhoA C-terminal peptide did not influence the spontaneous migration but blocked the SDF-1 induced migration. Each condition was analysed in duplicate.

Figure 7

Effect of pre-incubation with the Tat-PTD, Tat-Racl C-terminal peptide, Tat-Rac2 C-terminal peptide and Tat-RhoA C-terminal peptide on the Transwell migration of human mobilized peripheral blood cells in the absence (open bars) or presence (hatched bars) of 300 ng/ml Stromal Derived Factor 1 (SDF-1). None of the tested peptides affected the spontaneous migration. The SDF-1 induced migration was inhibited by the Tat-Racl C-terminal peptide, but not by the Tat-PTD or Tat-Rac2 C-terminal peptide. The Tat-RhoA C- terminal peptide reduced the SDF-1 induced migration by 50%. Each condition was analysed in duplicate. Figure 8

Effect of pre-incubation with the Tat-PTD, Tat-Racl C-terminal peptide, Tat-Rac2 C-terminal peptide and Tat-RhoA C-terminal peptide on the Transwell migration of human B-cell derived chronic lymphatic leukemia cells in the absence (open bars) or presence (hatched bars) of 100 ng/ml Stromal Derived Factor 1 (SDF-1). None of the tested peptides affected the spontaneous migration. However, the SDF- 1 induced migration was inhibited by the Tat-Racl C-terminal peptide in a concentration dependent manner. The Tat-PTD or Tat-RhoA C-terminal peptide did not influence the SDF-1 induced migration. Addition of the Tat-RhoA C-terminal peptide did not change the effect of the Tat-Racl C-terminal peptide on the migration of B- CLL cells. Each condition was analysed in duplicate.

Figure 9 Effect of pretreatment for 2 hours with 200 μg/ml of the Tat-Rac-1 C- terminal peptide on the morphological changes induced by the addition of 100 ng/ml SDF-1 to HL-60 cells, expressing GFP-actin. The cells were monitored by time-lapse confocal microscopy. Consecutive images, taken at time points 0", 15", 30", 60" show for the control marked responses in reorganization of the actin and large changes in overall cellular morphology. The cells, pretreated with the Tat-Rac- 1 C-terminal peptide did not show any clear response in either actin reorganization of morphology. This experiment is in line with the above data showing full inhibition of HL-60 cell migration upon pretreatment with the Tat-Rac-1 C-terminal peptide at this concentration.

Figure 10

NTH3T3 fibroblasts were grown on glass coverslips, serum starved for 18 hrs, preincubated with the indicated peptides, and stimulated with 10% fetal calf serum to induce contraction. The cells were monitored by time-lapse confocal microscopy. Consecutive images, taken at 0", 489" and 700" show clear cell contraction in the controls and the cells pretreated with the Tat- Rac-1 C-terminal peptide, but almost full inhibition of contraction in cells, incubated with the Tat-RhoA C-terminal peptide. This result shows the effectiveness of the Rho-inhibitory peptide. Moreover, the selectivity of the inhibition is underscored by the absence of effect of the Tat-Rac-1 C-terminal peptide.

Figure 11 NIH3T3 fibroblasts were incubated for 2 hrs with the indicated peptides, fixed, permeabilized and stained with anti-paxillin antibodies to visualize paxillin (green) and texas-red Phalloidin to visualize F-actin (red). The Tat- Rac-1 C-terminal peptide specifically induced a loss of paxillin staning from focal adhesions, that were detectable in the control cells and in the cell treated with any of the of the peptides, again underscoring the selectivity of the different peptides in their interference with intracellular events.

Figure 12

MDCK epithelial cells were treated with hepatocyte growth factor for 30 min to induce membrane ruffles in the absence or presence of the Tat-C- terminal peptides of Rac-1, Rac-2, RhoA and CDC42. Membrane ruffles (asterisks) were selectively inhibited by Tat-Rac-1 C-terminal peptide (arrow), underscoring the specificity of the inhibitory effects of these peptides.

DESCRIPTION OF THE INVENTION

The invention concerns a collection of cell-permeable, synthetic peptides that encode a protein transduction domain (PTD), linked to (a part of) the C- terminus of a Ras-like GTPase. These (fusion) peptides will enter any cell (eukaryotic cell, preferably mammalian cell) and will inhibit cellular functions, mediated by the GTPase of which the C-terminus was derived. Experiments using a set of these peptides encoding the C-termini of Rho-like GTPases show the potency and selectivity of inhibition, mediated by these peptides in a variety of primary and transformed human as well as rodent cell types. This invention will be useful to selectively interfere with signaling by Ras-like GTPases in vivo in order to counteract various types of human disease.

spacers A glycine residue may serve as a spacer in between the two domains. A spacer is not required, however. Whenever a spacer is used, it may comprise one or more amino acids, such as in particular 1 to 5 amino acids. A spacer may also comprise one or more -CH₂- moieties, optionally substituted by hydrophobic moieties (such as -CH₃ and -CδHs) or hydrophilic moieties (such as =O, -OH and -CH2OH) and optionally interrupted by or embraced by heteroatoms (such as -O-, -S- and -NH-). Suitable spacers can be, for example, a moiety having the formula -NH-(CH2)m-Q-(CH₂)n-CO- or a moiety having the formula -(CH2)m-Q-(CH2)n- wherein is optional and represents O, S, CO or NH, each of n and m may be an integer of from 0 to 10, preferably from 0 to 6, and m+n is an integer from 1 to 20, preferably from 1 to 12. Any divalent organic moiety may qualify as a suitable spacer and it is within the reach of a person skilled in the art to choose an appropriate linker moiety allowing the functional parts of the peptide to exhibit their intended function and optionally providing desirable characteristics to the peptide, such as higher or lower solubility, eg. in aqueous systems, higher or lower stability, eg. against proteases, etc.

Protein transduction domains

The amino acid sequences of PTDs that are preferably used for the fusion peptides of this invention are defined as follows: Antennapedia homeodomain, helix 3: RQILIWFQNRRMKWKK

Herpes Simplex Virus VP22: DAATATRGRSAASRPTERPRAPARSASRPRRPVE Human Immunodeficiency Virus-Tat: YGRKKRRQRRR Synthetic (artificial) PTDs: YARKARRQARR

YARAAARQARA YARAARRAARR

YARAARRAARA In view of their high efficiency, use of the HIV-Tat PTD or the synthetic PTD with the sequence: YARAAARQARA, is most preferred.

The invention is, however, not restricted to the use of these particular PTDs. Any amino acid sequence capable of penetrating (entering) into mammalian cells without being dependent on the use of a special carrier or receptor system may be used.

There are no particular restrictions on the length of the PTD sequences, but usually their length will be between about 10 and some 100 amino acids, more preferably between 10 or 11 and about 40 amino acids. In general, the shorter sequences are more preferable, i.e. those having from 11 to 20 amino acids.

GTPase C-terminal sequences Ras-like GTPases are strongly conserved in evolution and display high levels of homology. They occur in lower eukaryotes, like yeast and fungi, and in higher eukaryotes, like plants and animals, including humans. The present invention is applicable to all Ras-like GTPases, but those from the animal kingdom and particularly those from mammals are preferred, and most preferably those of human origin.

The C-terminal peptides of the different GTPases that are included in this invention are defined as follows. Within the majority of Ras-like GTPases, various alpha helices at homologous positions within the protein sequence can be identified. These helices determine, in combination with a series of beta-sheets, the overall three-dimensional structure. In addition, a large number of Ras-like GTPases contain the above-mentioned CAAX-box or a similar motif at their C-terminal end. The peptides that are in the list given in Table 1 span the region from the first amino acid following the 5^th and final alpha helix in the GTPase up until the amino acid directly preceding the cysteine residue of the CAAX box. Homology searches using the BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/) using Ras as a query, were used to define the position of the alpha helix in the Ras-homologues. In the case that no CAAX box could be identified, the peptides include the complete C-terminus. Distantly related Ras-like GTPases of which the homology was too low to define a peptide based on the above criteria, have not been included in the list and are therefore less preferred. This also includes the members of the Arf and Sar families.

Table 1: List of C-terminal sequences

Accession name/description amino acid sequence no.

Ras family

NP 005334 . 1 1 H-Ras Harvey rat sarcoma viral oncogene homolog QHKLRKLNPPDESGPGCMSCK

XP 001317 . 1 1 N-RAS viral (v-ras) oncogene homolog QYRMKKLNSSDDGTQGCMGLP

P01116 TRANSFORMING PROTEIN P21A (K-RAS 2A) (KI-RAS) (C- K-RAS) . QYRLKKISKEEKTPGCVKIKK

P01118 TRANSFORMING PROTEIN P21B (K-RAS 2B) (KI-RAS) (C- K-RAS) KHKEKMSKDGKKKKKKSKTK

NP 006261 . 1 1 R-RAS KYQEQELPPSPPSAPRKKGGGCP

NP 036351 . I I M-RAS QQIPEKSQKKKKKTK RGDRATGTHKLQ

XP 006102 . 1 1 oncogene TC21 (RRAS2) KFQEQECPPSPEPTRKEKDKKGCH

XP 039062 . 1 1 Rheb2, Ras homolog enriched in brain 2 KMDGAASQGKSS

XP 016528 . 1 1 ras-like protein LTPKKHTVKKRIGSRCINC

XP 002368 . 2 ] similar to ras-like protein LTPKKHTVKKKE

XP 040589. 1 1 ARHI : ras homolog gene family, member I KPTTGLQEPEKKSQMPNTTEKLLDKCIIM

XP_010646 novel Ras family protein AGIQYSDTQQQPKKSKSRTPDKMKNLSKSW KKY

NP 065078 . 1 1 KBRAS1 : I-kappa-B-interactmg Ras-like protein 1 QPQSKSSFPLPGRKNKGNSNSEN

P11233 RAL-A ARKMEDSKEKNGKKKRKS AKRIRER

P11234 RAL-B TKKMSENKDKNGKKSSKNKKSFKER

XP 044112 . 1 1 Ric RKEKEAVLAMEKKSKPKNSV KRLKSPFRKKKDSVT

AAB42214 Rin KKESMPSLMEKKLKRKDSL KKLKGSLKKKRENMT

CAA69175 RRP22: RAS-related on chromosome 22 VRARPAHPALRLQGALHPAR XP 052153.11 RAP1A KTPVEKKKPKKKS XP 052476.11 RAP1B RKTPVPGKARKKSS XP 007223.21 RAP2A MNYAAQPDKDDPC XP 003032.11 RAP2B YAAQSNGDEGCCSA XP 039483.11 VTS58635: ras-like protein VTS58635 GCARCKHVHAALRFQGALRRNR XP 007861.21 Rad LRRDSKEANARRQAGTRRRESLGKKAKRF GRIVARNSRKMAFRAKSKSCHDLSVL

Rab family

NP 004152.11 RAB1 KRMGPGATAGGAEKSNVKIQSTPVKQSGGG XP 035661.11 RAB1B KRMGPGAASGGERPNLKIDSTPVKPAGGG XP 037655.11 RAB2 KIQEGVFDINNEANGIKIGPQHAATNATHAGNQGGQQAGGG XP 038143.11 RAB3A EKMSESLDTADPAVTGAKQGPQLSDQQVPPHQD NP 002858.11 RAB3B DKMSDSLDTDPSM GSSKNTRLSDTPPLLQQN XP 009090.11 RAB3D EKMNESLEPSSSSGSNGKGPAVGDAPAPQPSS XP 034057.11 RAB4 NKIESGELDPERMGSGIQYGDAALRQ RSPRRAQAPNAQE

Table 1 (continued)

NP 057238. 1| RAB4b NKIDSGELDPERMGSGIQYGDASLRQLRQPRSAQAVAPQP

NP 004153 11 RAB5A KNEPQNPGANSARGRGVDLTEPTQPTRNQ

XP 006725 31 RAB5B KSEPQNLGGAAGRSRGVDLHEQSQQNKSQ

NP 004574 1| RAB5C KLPKNEPQNATGAPGRNRGVDLQENNPASRSQ

XP 006362 1| RAB6 QDRSREDMIDIKLEKPQEQPVSEGG

XP_ 039883 RAB6B GMENVQEKSKEGMIDIK DKPQEPPASEGG XP~^"031588 1| RAB7 KQETEVELYNEFPEPIKLDKNDRAKASAES

XP 050211 1| RAB7L RNSTEDIMSLSTQGDYIN QTKSSS S P 008925 41 RAB8 (c-mel) AKMDKKWKATAPGSNQGVKITPDQQKRSSFFR

XP 029546 1| RAB8B TKLNRKMNDSNSAGAGGPVKITENRSKKTSFFR

XP 010326 1| RAB9 ATEDRSDHLIQTDTVNLHRKPKPSSS

XP 010408 1| RAB9-lιke protein AVEEQLEHCMLGHTIDLNSGSKAGSS

XP 039752 11 RAB10 RKTPVKEPNSENVDISSGGGVTGWKSK

NP 004654 1| RAB11A RIVSQKQMSDRRENDMSPSNNVVPIHVPPTTENKPKVQ

XP 041617 1| RAB11B RIVSQKQIADRAAHDESPGNNVVDISVPPTTDGQKPNKLQ

NP 002861 1| RAB13 KSGGRRSGNGNKPPSTDLKTCDKKNTNK

XP 051616 1| RAB14 QNIQDGSLDLNAAESGVQHKPSAPQGGRLTSEPQPQREG

NP 071894 1| hypothetical protein FLJ1253S similar to RAB17 QRSDEEGQA RGDAAVALNKGPARQAK

XP 005820 41 RAB18 QTPGLWESENQNKGVKLSHREEGQGGGACGGY

XP 006829 1| Rab21 ETAQVDERAKGNGSSQPGTARRGVQIIDDEPQAQTSGGG

XP 030632 1| RAB22A STDANLPSGGKGFKLRRQPSEPKRS

XP 015946 1| RABL2B: SYKQNSQDFMDEIFQELENFSLEQEEEDVPDQEQSSSIETPSEEAASPHS

XP 004521 1| RAB23 QKLKQQIAEDPELTHSSSNKIGVFNTSGGSHSGQNSGTLNGGDVINLRPNKQRTKKNRNPFSS

XP 002080 1| RAB-25 (CATX-8 protein) QNSIRTNAITLGSAQAGQEPGPGEKRACCI

XP 007928 1| RAB26 QRSMKAPSEPRFR HDYVKREGRGAS

XP 046134 1| RAB27A KRMERCVDKS IPEGVVRSNGHASTDQLSEEKEKGA

NP 004154 21 RAB27B KRMEQCVEKTQIPDTVNGGNSGNLDGEKPPEKK

XP 003581 11 RAB28 GIKLNKAEIEQSQRIVRAEIVKYPEEENQHTTSTQSRI

XP 006025 21 RAB30 QNTLVNNVSSPLPGEGKSISYLT

NP 006859 1| RAB31 PLDPHENGNNGTIKVEKPTMQASRR

XP 004076 11 RAB32 VNHQSFPNEENDVDKIKLDQETLRAENKSQ

XP 017932 1| RAB33A QGKVQKLEFPQEANSKTSCP

NP 112586 1| RAB33b SHKPLMLSQPPDNGIILKPEPKPAMT

XP 007037 21 RAB35 RAKKDNLAKQQQQQQNDVVKLTKNSKRKKR

XP 037054 1| RAB36 FEQSV QDLERQSSARLQVGNGDLIQMEGSPPETQESKRPSS G

XP 015771 1| RAB38 ANECDLMESIEPDVVKPHLTSTKVASCSG

XP 031429 1| RAB39 ANVLAELEKSGARRIGDVVRINSDDSNLYLTASKKKPTCCP

Table 1 (continued)

Rho family

XP 047561.11 RhoA LQARRGKKKSG XP 039133.11 RhoB QKRYGSQNGCINC XP 052125.11 RhoC QVRKNKRRRG NP 055393.11 RhoD RNFWRRITQGF XP 008210.11 Rho7 RGHRQLRRTDSRRGMQRSAQLSGRPDRGNEGEIHKDRAKS XP 035512.11 RhoE (Rho8) KTNKNVKRNKSQRATKRISHMPSRPELSAVATDLRKDKAKS XP 006153.31 RhoG LNPTPIKRGRS XP 047535.11 RhoH RRNRRRLFSINE

095661 RhoI KPTTGLQEPEKKSQMPNTTEKLLDK

XP 005353 . 2 1 hypothetical protein DKFZp761C07121 RRTVSLQIDGKKSKQQKRKEK KGK

Q9HBH0 RIF VALSALKKAQRQKKRRL

XP 016309 . 1 1 Racl CPPPVKKRKRK

XP 038672 . 1 1 Rac2 CPQPTRQQKRA

NP 005043 . 1 1 Rac3 CPPPVKKPGKK

XP 032917 . 1 1 CDC42 EPPEPKKSRR

XP 046971 . 1 1 WRCH-1: nt-1 responsive Cdc42 homolog YSDTQQQPKKSKSRTPDKMKNLSKS WKKY

P17081 TC10 AILTPKKHTVKKRIGSRCINC

XP 050746. 1 1 TCL, TClO-like Rho GTPase HPKKKKKRCSEGHSC

Ran family

XP 012170 Ran DPNLEFVAMPALAPPEWMDPALAAQYEHDLEVAQTTALPDEDDDL

Other Ras-like GTPases

XP 029564.11 hypothetical protem similar to small G proteins YSSLPEKQDQCCTT XP 032506.11 49777 RKTPVPGKARKKSS XP 041103.11 hypothetical protem MGC15754 RRRMVQGKTRRRSSTTHVKQAINKMLTKISS XP 017749.11 hypothetical protein MGC2827 SHKQQPSΞTPEKRRTSLIPRPKSPNMQDLKRRFKQALSAKVRTVTSV NP 055125.11 similar to mouse Ras, dexamethasone-induced 1; tumor endothelial marker 2 KLPHEMSPALHRKISVQYGDAFHPRPFCMRRVKEMDAYGMVSPFARRPSVNSDLKYIKAKVLREGQARERDK

NP 057168.11 ras-related protein KLPSEMSPDLHRKVSVQYCDVLHKKALRNKKLLRAGSGGGGGDPGDAFGIVAPFARRPSVHSDLMYIREKASAGSQAKDKER XP 005162.11 GEM, GTP-binding protein overexpressed in skeletal muscle LRRDSKEKNERRLAYQKRKESMPRKARRFWGKIVAKNNKNMAFKLKSKSCHDLSVL

XP 046159.11 GES; REM protein LRRRDSAAKEPPAPRRPASLAQRARRFLARLTARSARRRALKARSKSCHNLAVL XP 007618.21 Ris RELEKSPLTRPLFISEERALPHQAPLTARHGLASCTFNTLSTINLKEMPTVAQAKLVTVKSSRAQSKRKAPTLTLLKGFKIF

The invention is, however, not restricted to the use of the particular sequences shown in Table 1. Any amino acid sequence capable of inhibiting signaling by a Ras-like GTPase may be used.

There are no particular restrictions on the length of the Ras-like GTPase signaling inhibiting sequences, but usually their length will be between about 10 and some 150 amino acids, more preferably between 10 or 11 and about 100 amino acids. In general, the shorter sequences are more preferable, i.e. those having from 11 to 40 or even between about 11 and 20 amino acids.

The peptides from the different GTPases may include from 1 to 5 additional amino acids preceding the sequences depicted herein (i.e. upstream into the homologous fifth alpha helix) to improve functionality and specificity.

Moreover, a lipid moiety, such as eg. palmitoyl or myristoyl, can be covalently linked to one or more of the amino acids of the peptide construct, in particular to the amino terminal amino acid of the PTD-fusion peptide or to the GTP-ase C-terminal peptides themselves (i.e. without the PTD domain). Such lipid moieties have previously been used to bring synthetic, protein kinase C-activating peptides into cells (Eichholtz et al., 1993). The addition of such lipids to a PTD-fusion peptide may promote localization of the peptide to cellular membrane, which may enhance specificity and potency of the peptide. The position of the constituent domains of the peptides of the invention is not particularly restricted. The amino acid sequence corresponding to a protein transduction domain (PTD) which has cell-penetrating capacity may be at the N-terminal end of the peptide, at the C-terminal end of the peptide, or even be enclosed between neighbouring sequences. Similarly, the amino acid sequence corresponding to a variable part of the C-terminus of a Ras-like GTPase which has Ras-like GTPase signaling inhibiting capacity may be at the C-terminal end of the peptide, at the N-terminal end of the peptide, or be enclosed between neighbouring sequences. The preferred position of the constituent domains is however the amino acid sequence corresponding to a protein transduction domain (PTD) which has cell-penetrating capacity at the N-terminal end of the peptide and the amino acid sequence corresponding to a variable part of the C-terminus of a Ras-like GTPase which has Ras-like GTPase signaling inhibiting capacity at the C-terminal end of the peptide. Applications

This invention is primarily designed to inhibit excessive and uncontrolled cellular signaling, as generally occurs in many types of disease. This uncontrolled signaling involves events that are for the most part downstream of Ras-like GTPases, including cell growth and division, dissociation and migration of tumor cells, migration of activated immune cells, and apoptosis. The human diseases that are putative therapeutic targets for these fusion peptides represent a wide range, due to the fact that the Ras-like GTPases are crucial in many physiological processes.

The present invention represents an important addition to the various types of drugs that are currently available or are under investigation, since this invention is based on the combination of a cell-penetrating domain and a specific peptide sequence. This is explained in more detail below using cancer as an example.

In addition to established means to treat cancer, such as chemotherapy or radio-therapy, there are at the moment various types of compound in different stages of clinical trials that have promising anti-cancer features. These include: • Antibodies to the extracellular regions of specific growth factor receptors that show increased expression on certain types of tumor, e.g. ErbB2 on metastatic breast cancer cells;

• Anti-sense oligonucleotides that are used to reduce expression of proteins that are required in growth factor signaling, e.g. the protein kinase Raf. This technique has not yet found wide application;

• Inhibitors of transcription factors, such as steroids or derivatives thereof: these compounds enter cells freely;

• Small molecules or peptidomimetics that are designed to specifically interfere with the activity of particular signaling proteins, such as receptor- or non-receptor tyrosine kinases. These molecules are usually modelled on the basis of crystallography data to block, for instance, ATP binding, and are usually sufficiently small and hydrophobic to enter cells rapidly;

• Small molecule -inhibitors of farnesyl or geranylgeranyl transferases (FTIs or GGTIs): during synthesis, these lipid anchors are added to the

C-termini of small GTPases and the inhibition of their synthesis has proven very promising in anti-cancer treatment. Drawbacks are that the exact target of the FTIs and GGTIs is not fully clarified, the inhibition is not specific for a certain GTPase, and lipid modification can be altered, resulting in a GTPase with a different lipid modification, which still localizes to the (plasma) membrane and thus is still active. In this overview of recent anti-cancer drugs, larger peptides (>5 amino acids) are lacking. The use of such peptides (ranging from 10 amino acids upwards, such as up to 40 or more preferably up to about 25 amino acids) would greatly favor specific interference with protein-protein interactions. The reason that such peptides can hardly be used as therapeutic agents is that the majority (if not all) of these will not spontaneously enter cells. By now providing specific antagonistic peptides with cell-penetrating capacity, it will become feasible to specifically and efficiently interfere with intracellular signaling in human disease. An additional advantage of the use of peptides as therapeutic agents is that the immune system does not mount an efficient immune response to relatively short linear peptides. Moreover, a significant part of these peptides contains sequences that will be recognized as 'self and thus will not trigger massive immune activation.

Functions of the Ras-like GTPases As described above, the Ras-like GTPases can be subdivided into different families (Takai et al., 2001). These protein families are generally associated with distinct cellular functions, although extensive crosstalk between, for instance, Ras and Rac signaling pathways has also been described. To clarify the possible effects and underscore the putative applications of the different PTD-GTPase C-termini peptides mentioned in this document, a brief overview of the main functions of the relevant GTPase families is provided here.

The Ras-like GTPases have an essential role in cell growth and division and in the modulation of gene expression. Ras is the prototypic GTPase that is known to activate the Map-kinase pathway, which is of paramount importance in the onset of cell division. The RaplA GTPase was originally described as a protein (K-revl) that reverted the morphology of Ki-Ras-transformed cells and has recently been described to be required for activation of integrins in leukocytes. In addition, Rapl also activates the Map kinase pathway in neuronal cells, which is important for neuronal differentiation. The Ral proteins play a role in endocytosis of growth factor receptors as well as in exocytosis of, for instance, Von Willebrand Factor (Takai et al., 2001; De Leeuw et al., 2001), suggesting that the Ral proteins are important in inflammation and coagulation.

The Rho-like GTPases are principally involved in the orchestration of the actin cytoskeleton during cell adhesion, spreading and migration (Hall, 1998). As a result they are instrumental during tumor cell metastasis (Price and

Collard, 2001). In addition, Rho proteins are involved in the control of cell-cell adhesion in epithelial and endothelial cells (Kaibuchi et al., 1999). The Rac GTPases (in particular Rac2) are well known for their activation of the NADPH oxidase complex in human neutrophils. Recent data indicate that Rac also is involved in the production of reactive oxygen species in cells, other than neutrophils, such as endothelial cells. Given the damage that can occur due to endothelial reactive oxygen production during, for instance, atherosclerosis and other inflammatory conditions, this represents an important effect of Rac signaling. Rho proteins also activate kinase pathways (such as the JNK or the p38 MAP kinase pathway) that are implicated in the regulation of gene expression and cell division.

The Rab GTPases are well known for their role in vesicular trafficking and exocytosis (Takai et al., 2001). The different members of the family show differential localization in the cell and are involved in various events that are required for intracellular vesicle transport, for instance in neuronal cells. A number of Rab proteins are associated with tumor formation, especially in melanomas, but also in adenomas and adenocarcinomas. The Rab6c protein has recently been implicated in drug resistance of mammary tumor cells (Shan et al., 2000), suggesting that inhibition of vesicular transport might potentiate the effects of anti-cancer drugs. In addition, Rab proteins have been implicated in the organization of the (tubular) cytoskeleton and in cell migration.

The Ran GTPase is involved in nuclear transport of RNA and protein, cell cycle regulation at the Gl/S interphase, chromatin decondensation after mitosis and chromosome stability. Given this function, inhibition of the protein may also result in impaired cell division.

The PTD-GTPase fusion peptides can be used either as a single peptide or as a mixture of 2 or more peptides to reduce or block a variety of intracellular signaling events that are functionally relevant in human diseases. Because of their apparent specificity, these peptides can be combined with existing therapies. Based on the recently obtained experimental data, these peptides are predicted to interfere with the following pathophysiological conditions: • uncontrolled cell division and malignant growth;

• dissociation of cells from solid tumors; • tumor cell metastasis;

• angiogenesis and tumor growth due to inhibition of migration of endothelial cells;

• activation of immune cells, for example of granulocytes;

• influx of activated immune cells in tissue such as lung or in joints; • modulation of apoptosis; or a combination of these.

The range of malignancies/disorders for which these peptides may have clinical relevance include:

Cancer: Different types of solid tumors such as carcinomas, sarcomas and melanomas, with high as well as low metastasizing capacity; non-solid tumors such as leukemias and lymphomas of different origin and the like.

The Ras GTPase is an essential mediator for cell division in normal as well as in transformed cells. Many forms of cancer have mutations in the p21Ras oncogene, representing a major event in the development of the disease. Even if Ras is not mutated, its activity is required for tumor growth, as tumor development obviously depends on efficient cell division. Thus, inhibition of Ras signaling will be effective in reducing growth of any type of malignant cell. Although for Rho-like GTPases, no mutations have so far been found in human tumors, their activity is required for tumor cell metastasis (Michiels et al., 1995) and reduced adhesion and inhibition of Rac/Rho signaling will therefore impair tumor cell migration, dislodge me nt from solid tumors and metastasis. In tumor cells, high levels of reactive oxygen species (ROS) can be found. These can also play an important role in cell dissociation and metatasis, for instance in tumors of epithelial origin. The production of ROS is mediated by a family of (recently identified) oxidases, of which relatively little is known. However, considerable evidence has shown that Ras and Rac are important for activating the ROS-generating enzymes in various cell types and inhibition of either of these (or both) will therefore further reduce disease progression. In addition, Rac signaling is involved in the formation of new blood vessels (angiogenesis) and is therefore crucially involved in secondary tumor formation.

Inflammatory and proliferative diseases. Chronic as well as acute inflammatory diseases including: Rheumatoid Arthritis, Multiple Sclerosis, Psoriasis, Asthma, and the like. Inflammatory as well as proliferative diseases such as psoriasis are accompanied by excessive and uncontrolled influx into tissues of granulocytes and activated T- and/or B-cells, which can be a major cause of the tissue damage that is associated with the disease. Inhibition of this migration by the fusion peptides encoding sequences from the Rho-like GTPases will therefore help to prevent the damage that is associated with these diseases to tissues such as lung, skin, joints and bone.

Atherosclerosis. Since atheroclerosis is in essence an inflammatory disease, also here inhibition of migration of activated immune cells, primarily monocytes, across the inflamed vessel wall will be important to reduce or block disease progression. Moreover, development of atherosclerosis has been associated with the production of ROS in endothelial cells. The inhibition of this event, through inhibition of Rac signaling, will therefore have additional beneficial effects in reducing the levels of intracellularly produced ROS and the concomitant endothelial damage.

Other diseases. These include choroideremia, which is linked to a mutation in a Rab-binding protein and is associated with disturbed Rab functioning and, for instance, Alzheimer's disease, which is associated with increased signaling by p21Ras resulting in dedifferentiation of neuronal cells and apoptosis (Gartner et al., 1999). Also increased activation of an Vav, an exchange factor for Rac/Rho/CDC42 in T-cells, has been associated with increased susceptibilty of mice to experimental autoimmune encephalomyelitis, suggesting that inhibition of these GTPases might be beneficial in modulating T-cell reactivity in multiple sclerosis (Chiang et al., 2000).

Administration of peptides according to the invention The means by which the fusion peptides of the invention can be applied in vivo are: by intravenous or intraperitoneal injection of the peptides, dissolved in physiological saline; local injection, for instance into a solid tumor and its surrounding tissue; as an aerosol, in which the peptide(s) can be inhaled, for instance for treatment of asthma; orally, where the resistance of a particular peptide to cleavage by enzymes in the digestive system needs to be assessed. The concentrations that are required for effective treatment are in the micromolar range. Based on the in vitro data, the peptides (c.q. their effects) are relatively stable (>40 hrs), however, the stability of each peptide will differ, depending on its sequence, and will require careful assessment for in vivo application. The toxicity of the Tat PTD in mice is low or absent (Schwarze et al., 1999), although higher concentrations than used in this work may require reassessment. The toxicity level of the GTPase-fusion peptides will require careful assessment. It is considered to be within the reach of a skilled person to determine the optimal administration route and administration form, suitable pharmaceutically acceptable carriers or diluents, suitable dosage and dosage regime, etc., considering various factors of possible relevance, such as condition, age and/or weight of the patient, severity of the illness in question, and others.

The invention will be illustrated by some examples that are merely given for illustrative purposes and should therefore not be construed as restricting the scope of the invention.

EXAMPLES

Fusion peptides

The work that has formed the basis for this invention has focussed on the subfamily of Rho-like GTPases, primarily Racl, Rac2, RhoA and CDC42. The C-terminal sequences of Racl, Rac2, RhoA and CDC42 were chemically synthesized in combination with the PTD, derived from HIV Tat. The peptides were used in most of the in vitro experiments in a concentration of 200 μg/ml (approximately 70 μM). The data obtained using these peptides in a variety of cell systems and assays are represented in the attached figures. The complete sequences of these peptides as used in the experiments shown are:

Tat-Racl C-terminal peptide : YGRKKRRQRRRGCPPPVKKRKRK

Tat-Rac2 C-terminal peptide: YGRKKRRQRRRGCPQPTRQQKRA

Tat-RhoA C-terminal peptide: YGRKKKRQRRRGLQARRGKKKSG Tat-CDC42 C-terminal peptide: YGRKKRRQRRRG LEPPEPKKSRR The methods applied in the different types of in vitro assays that are used in the analysis of these peptides are described below.

Assays and results

The analysis of the above mentioned peptides has focussed on the in vitro migration of primary human leukemic cells as well as established leukemic cell lines. It is well known that (leukocyte) migration, be it either spontaneous or driven by chemotaxis, is, to a large extent, dependent on signaling by the Rac GTPase. The goal of the present analysis was to test the hypothesis that the Racl-C-terminal fusion peptide would act as an inhibitor of leukemic cell migration. Moreover, the selectivity of the inhibition was established.

Analysis of migration The HL60 and the KGlA cell lines were obtained from ATCC (Rockville,

MO) and maintained in Iscove's Modified Dulbecco's Medium (IMDM; BioWhittaker, Brussel, Belgium) containing L-glutamine, penicillin 100 U/ml, streptomycin 100 μg/ml, β-mercapto-ethanol (0.1%) and 10% fetal calf serum (Gibco, Life technologies, Paisley, Scotland). Cord Blood (CB) was collected after delivery, according to the guidelines of

Eurocord Nederland. Mobilized Peripheral blood (MPB) progenitor cells were obtained from patients treated with chemotherapy and G-CSF to induce stem cell mobilization. MPB and CB cells were processed using a ficoll- gradient (1.077 g/cm²; Pharmacia Biotech, Uppsala, Sweden). Mononuclear cells were harvested and resuspended in PBE buffer, containing PBS, 0.5% bovine serum albumine (BSA) and 5mM EDTA.

CD34⁺ cells from CB and MPB were isolated with the Macs-selection system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The purity was determined using flowcytometry and ranged from 94-98%.

Migration assays were performed in Transwell plates (Costar, Cambridge, MA) of 6.5 mm diameter with 5 μm pore filters. The upper and the lower compartment of the Transwells were separated by a filter coated overnight at 4°C with bovine fibronectin (Sigma, St Louis, MO) at a concentration of 20 μg/ml in PBS. Before adding the cells to the upper compartment, the coated Transwells were washed twice with assay medium (IMDM with 0.25% BSA (fraction V; Sigma). Before use the cells were washed twice in assay medium. The cells, in a concentration of 1 xlO⁶ nucleated cells/ml were incubated with various peptides coupled to the Tat PTD. Two periods of incubation were applied. First, a pre-incubation lasting for 30 minutes at 37°C and 10% CO2. After the pre-incubation, the cells were washed with assay medium and again brought to a concentration of 1 xlO⁶ cells/ml. Secondly, the Tat-peptides were present during the pre-incubation as well as during the migration assay, i.e. for a total of 4.5 hours.

To perform the migration assay, 100,000 cells in 0.1 ml assay medium were added to the upper compartment and 0.6 ml of assay medium with or without Stromal Derived Factor-lα (SDF-1) was added to the lower compartment. SDF-1 was purchased from Strathmann Biotech GmbH (Hannover, Germany). An 0.1 ml sample of cells in assay medium was diluted with 0.5 ml assay medium and was kept as an input control for quantitation of the number of migrated cells. The Transwell plates were incubated at 37°C, 5% CO₂, for 4 hours. Cells that had migrated to the lower compartment were collected in a tube to which a fixed number of control cells, i.e. HL60 or KGlA cells, labeled with Calcein AM (Molecular Probes, Leiden, The Netherlands) were added. The control cells were added to the tubes just before the analysis. Flow- cytometrical analysis was used to determine the ratio between labeled cells and unlabeled cells. By comparing this ratio to the input control, the number of migrated cells was quantitated.

Analysis of the migration of various types of human cell (Human leukemic cell lines, primary hematopoietic stem cells i.e. CD34⁺ cells, B-cell derived chronic lymphatic leukemia cells) following (pre)treatment with the different fusion peptides show that the peptides are selective in their inhibitory effects (see figs 2-8). The Tat-Racl C-terminal peptide inhibits migration of leukocytes (primary as well as immortalized as well as malignant) in the concentration range of 20-200 μg/ml. The Tat-RhoA C-terminal peptide was 10-20 fold less effective in inhibiting cell migration. Moreover, the peptides are most effective when present for prolonged periods (i.e. during the in vitro migration assay) since preincubation of the peptides, followed by washing, reduced their inhibitory capacities by at least 10-fold. In these assays, the Tat-Rac2 C- terminal peptide was also included. Despite the similarity of the Racl and Rac2 peptides, the Tat-Rac2 C-terminal peptide, in the majority of the experiments did not interfere with spontaneous or SDF-induced cell migration. Parallel analysis of human neutrophil migration in response to the chemokine Interleukin-8 revealed that 5-10 fold higher concentrations of the Tat-Rac-1 C-terminal peptide were required to reduce migration. This may be caused by the extremely high levels of Rac signaling in the very motile neutrophils, but may also relate to proteolytic breakdown of the peptides at the neutrophil external cell surface.

The Tat-Racl C-terminal peptide (but not its control) also inhibited chemokine-induced actin polymerization in HL-60 cells. This rapid response is a hallmark for chemokine signaling and is directly related to the cell's migratory capacities. This inhibition explains why the various cells are impaired in their migratory response in the abovementioned transmigration assays by the Tat-Racl C-terminal peptide.

Microscopical analysis of HL60 cells Complementary analysis using time -lapse confocal laser scanning microscopy of HL-60 cells expressing a Green fluorescent protein(GFP)-actin fusion protein (Voermans et al., 2001) showed that addition of 100 ng/ml SDF induced a clear morphological response and a redistribution of the cellular actin in a fraction of the cells which was completely absent in cells that were pretreated with the Tat-Racl C-terminal peptide (200 μg/ml) (Fig. 9). This result is in agreement with the data that show the inhibition of migration of cells following pretreatment of the Tat-Racl C-terminal peptide since this pretreatment apparently precludes chemokine-induced cytoskeletal changes that are crucial for proper migration.

Analysis of cellular contraction

Analysis of serum-induced contractility of fibroblasts is an established response, largely dependent on signaling by the Rho GTPase. In these assays, murine NIH3T3 fibroblasts were used as a model. The cells were cultured in serum-free medium for 18 hrs, prior to the experiment. Preincubation with either the Tat-Racl C-terminal peptide or the Tat-RhoA C-terminal peptide did not affect the normal cellular morphology under resting conditions as judged by phase-contrast microscopy. However, the pronounced cellular contraction, induced by the addition of 10% fetal calf serum, was almost completely inhibited in cells, pretreated with the Tat-RhoA C-terminal peptide (Fig. 10). The Tat-Racl C-terminal peptide had a minor effect on the contraction, induced by serum. These data, in combination with the results from the migration assays, indicate the effectivity as well as the selectivity of the different peptides in the various assays and cell types.

Analysis of focal adhesions

In these assays, the presence and distribution of focal adhesions in NIH3T3 fibroblasts was analysed. Focal adhesions are formed by clustering of integrins, inte grin-binding proteins and the actin cytoskeleton and form an important structure that regulates the adhesion of cells to the extracellular matrix. Focal adhesions can be clearly visualized using immunocytochemistry for a number of proteins, such as vinculin or paxillin. The paxillin distribution in focal adhesions following a 2 hrs treatment of NIH3T3 cells with the different above-mentioned peptides was analysed using immunocytochemistry in combination with confocal microscopy (Fig. 11). The Tat-Racl- C terminal peptide specifically induced a loss of paxillin staining in cellular extensions (e.g. lamellipodia). In contrast, the other three peptides left the paxillin distribution at the ends of actin stress fibers (focal adhesions) intact. This assay in particular reveals the marked specificity of these peptides.

Functionality of the fusion peptides

Although the use of dominant negative (i.e. inhibitory) variants of > GTPases has been reported (e.g. Soga et al, 2001), this involved the use of a cell-permeable version of a full-length recombinant protein. The synthesis, purification and application of these proteins is much more complex and the results obtained are more prone to variabihty than the synthetic fusion peptides described here. This also means that the functionality of these large proteins is always difficult to assess. The collected data discussed above allow two main conclusions to be drawn. Firstly, the data show that the peptides are indeed effective in inhibiting cellular responses that are known, based on a variety of other approaches and described in literature, to be dependent on the activity of one (or more) particular GTPase(s). Secondly, in most assays, the peptides show remarkable specificity. This indicates first of all that cellular toxicity or an overall impairment of signaling pathways is not a general feature of these peptides and that the peptides are attractive tools in inhibiting specific cell functions, not only in vitro, but perhaps also in vivo. References

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Claims

Claims

1. A peptide comprising an amino acid sequence corresponding to a protein transduction domain (PTD) which has cell-penetrating capacity and an amino acid sequence corresponding to a variable part of the C-terminus of a Ras-like GTPase which has Ras-like GTPase signaling inhibiting capacity.

2. A peptide of claim 1, wherein said amino acid sequence corresponding to a PTD is selected from the group consisting of (a) a PTD of the Tat-peptide of Human Immunodeficiency Virus; (b) a PTD of the homeodomain protein of Antennapedia; (c) a PTD of the Vsp22 protein of Herpes Simplex Virus; and (d) an artificial PTD with cell-penetrating capacity.

3. A peptide of claim 2, wherein said PTD is a HIV Tat-PTD with amino acid sequence YGRKKRR RRR.

4. A peptide of claim 2, wherein said PTD is an Antennapedia homeo- domain-PTD with amino acid sequence R ILIWFQNRRMKWKK.

5. A peptide of claim 2, wherein said PTD is a HSV Vsp22-PTD with amino acid sequence DAATATRGRSAASRPTERPRAPARSASRPRRPVE.

6. A peptide of claim 2, wherein said PTD is an artificial PTD with amino acid sequence selected from the group consisting of (a) YARKARR ARR; (b) YARAAARQARA, (c) YARAARRAARR, and (d) YARAARRAARA.

7. A peptide of claim 1, wherein said amino acid sequence corresponding to a variable part of the C-terminus of a Ras-like GTPase which has Ras-like GTPase signaling inhibiting capacity is an amino acid sequence from a Ras- like GTPase selected from the group consisting of (a) GTPase of the Ras family, (b) GTPase of the Rab family, (c) GTPase of the Rho family, (d) GTPase of the Ran family, and (e) another Ras-like GTPase.

8. A peptide of claim 7, wherein said amino acid sequence is selected from the group consisting of the amino acid sequences shown in Table 1.

9. A peptide of claim 8, wherein said amino acid sequence is selected from the group consisting of (a) Racl sequence CPPPVKKRKKK; (b) Rac2 sequence CPQPTRQQKRA; (c) RhoA sequence LQARRGKKKSG; and (d) CDC42 sequence LEPPEPKKSRR.

10. A peptide of claim 1, wherein said protein transduction domain (1) and amino acid sequence (2) are connected to eachother through a spacer.

11. A peptide of claim 10, wherein said spacer comprises one or more, preferably from 1 to 5 amino acids.

12. A peptide of claim 10, wherein said spacer is a glycine residue.

13. A peptide of claim 1, wherein said amino acid sequence corresponding to a PTD comprises from 10 to 100, preferably from 10 or 11 to about 40 amino acids and said amino acid sequence corresponding to a variable part of the C- terminus of a Ras-like GTPase comprises from 10 to 150, preferably from 10 or 11 to about 100 amino acids.

14. A peptide of claim 1, which is selected from the group consisting of (1) the Tat-Racl construct: YGRKKRRQRRRGCPPPVKKRKRK;

(2) the Tat-Rac2 construct: YGRKKRRQRRRGCPQPTRQQKRA;

(3) the Tat-RhoA construct: YGRKKRRQRRRGLQARRGKKKSG; and

(4) the Tat-CDC42 construct: YGRKKRRQRRRGLEPPEPKKSRR.

15. A peptide of claim 1, which furthermore contains one or more lipid moieties attached to an amino acid residue.

16. A peptide of any one of claims 1 to 15, for use in a method to inhibit cellular functions mediated by the said Ras-like GTPase.

17. A peptide of claim 1 for inhibiting leukemic cell migration, wherein said peptide is Tat-Racl construct YGRKKRRQRRRGCPPPVKKRKRK.

18. A method for manufacturing an inhibitor of a Ras-like GTPase, comprising analyzing said Ras-like GTPase to determine the amino acid sequence located between the final alpha helix, counting from the N- to the C- terminus, and the CAAX-box or similar motif, if present, or the C-terminus itself when no CAAX-box or similar motif is present, selecting an amino acid sequence corresponding to a natural or artificial protein transduction domain (PTD), and producing a peptide as defined in any one of claims 1-15 comprising said selected amino acid sequence corresponding to a PTD, which has cell- penetrating capacity, said determined amino acid sequence corresponding to a variable part of the C-terminus of said Ras-like GTPase, which has capacity to inhibit signaling by said Ras-like GTPase, and optionally a spacer between both amino acid sequences.

19. A method for inhibiting cellular functions mediated by a Ras-like GTPase in eukaryotic cells, preferably mammalian cells, comprising contacting said cells with a peptide as defined in any one of claims 1-15.

20. A pharmaceutical composition comprising a peptide as defined in any one of claims 1-15 together with a pharmaceutically acceptable carrier or diluent.

21. A method of treatment of a mammal, in particular a human being, in need of a treatment that inhibits cellular functions mediated by a Ras-like

GTPase, comprising administering to said mammal a peptide as defined in any one of claims 1-15 in an effective dose to inhibit the cellular functions of said Ras-like GTPase.