WO2003083441A2

WO2003083441A2 - Identification of apoptotic peptides and methods of use thereof

Info

Publication number: WO2003083441A2
Application number: PCT/US2003/009748
Authority: WO
Inventors: Gabriel Del Rio Guerra; Dale E. Bredesen
Original assignee: Buck Institute
Priority date: 2002-03-29
Filing date: 2003-03-28
Publication date: 2003-10-09
Also published as: AU2003231980A1; WO2003083441A3; AU2003231980A8

Abstract

Invention methods provide a computational method (APAP) to detect short potential apoptotic (PAPs). In preferred embodiments invention methods are based on the prediction of the helical content of peptides, the hydrophobic moment, and the isoelectric point (IP). PAPs are toxic against bacteria and mitochondria, but not against mammalian cells when applied extracellularly. Using invention methods, peptides such as Substance (SP) were identified as a PAP and subsequently used to induce apoptosis. Accordingly, invention methods allow for the detection of apoptotic peptides and the induction of apoptoisis in target cells. Such methods are useful in cells such as cancer cells, for example, using pro-apoptotic peptides for chemotherapy.

Description

IDENTIFICATION OF APOPTOTIC PEPTIDES AND METHODS F USE THEREOF

FIELD OF THE INVENTION

The present invention relates to apoptosis. In a particular aspect, the invention relates to the identification and use of apoptotic peptides.

BACKGROUND OF THE INVENTION

An antibacterial peptide, when targeted intracellularly to the angiogenic vasculature (i.e., to the endothelial cells) supplying tumors, can induce apoptosis by swelling their mitochondria, (Ellerby et al, Nat. Med. 5:1032-8, 1999), leading to the loss of tumor blood supply and consequent tumor regression. These chemotherapeutic peptides are known as homing pro-apoptotic peptides. The pro-apoptotic part of the peptides is designed to induce endothelial cell apoptosis through mitochondrial swelling. The peptides are positively charged and the mitochondria, like bacteria, have negatively charged membranes, thus the peptides are attracted to and disrupt the mitochondrial membrane (Oren and Shai, Biopolymers 47:451-63, 1998; Matsuzaki et al, Biochemistry 34:3423-9, 1995). Initial pro-apoptotic peptides were modeled with a 21- residue peptide, of which the carboxy terminal 14 amino acids represented the pro- apoptotic peptide, with the amino terminal 7 amino acids comprising the targeting peptide and a glycinylglycine bridge. The therapeutic index (TI) of the model pro- apoptotic peptide is approximately 10.

Apoptosis in mammals and other eukaryotic organisms is a characteristic process of cell death, which can, among its other effects, limit the spread of viruses and other intracellular organisms (Hershberger et al, J. Virol. 66:5525-33, 1992). For example, the difference in viral titer during Baculoviral infection with and without apoptosis inhibition is 200-15,000-fold (Hershberger et al, supra). Thus, apoptosis is a mechanism of defense against pathogenic infections.

Apoptosis proceeds by the activation of a group of cysteine proteases called caspases (Salvesen and Dixit, Cell 91:443-6, 1997). One of these, caspase-9, is activated when cytochrome c is released from mitochondria, which may occur with the disruption of the mitochondrial outer membrane (Zou et al, J. Biol. Chem. 274:11549-56, 1999). This cytochrome c release in apoptotic cells may be induced by pro-apoptotic members of the Bcl-2 family, such as Bax and Bid, although the mechanism by which this is achieved is incompletely understood (Jurgensmeier et al, Proc. Natl. Acad. Sci USA 95:4997-5002, 1998). Nonetheless, the similarities between bacterial and mitochondrial membranes (and membrane potentials) suggested the possibility that there may be similarities between the effect of the antibacterial/ pro-apoptotic peptides and pro-apoptotic Bcl-2 family members.

Antibacterial peptides in multicellular organisms are thought to serve as a defense against microbial pathogens. Originally found in invertebrates, antibacterial peptides have now been described in humans and many other organisms (Oren and Shai, supra). Among these peptides, the most well characterized are the short linear peptides (less than 40 amino acids in length) that do not contain cysteine residues. A characteristic shared by virtually all of these peptides is the presence of an amphipathic α-helical structure, which stabilizes in environments of hydrophobic nature (Javadpour et al, }. Med. Chem. 39:3107-13, 1996) (although this helical structure has been shown not to be necessary for membrane lysis produced by a truncated form of pardaxin, an antibacterial peptide from the sole Pardachirus marmoratus (Oren et al, Eur. J. Biochem. 259:360-9, 1999). Another characteristic shared by some of these peptides is selectivity, in that membranes from bacteria are targeted by these peptides more efficiently than mammalian plasma membranes. This selectivity is based on the complementary charge between the peptides, which are characteristically positively charged, and the negatively charged membranes of bacteria (Oren and Shai, supra; Matsuzaki et al, supra).

Structurally, these peptides typically adopt an unfolded conformation in aqueous solution. On contact with a membrane with a complementary charge, these peptides anchor to the membrane and assume an α-helical conformation. In that conformation, these peptides would either he over the membrane surface in a carpet-like arrangement (in which the peptide backbone lies parallel to the membrane), or penetrate it according to the barrel-stave mechanism (in which the peptide backbone lies perpendicular to the membrane) (Oren and Shai, supra). In either case, the integrity of the membrane would be disturbed, eventually leading to membrane lysis. There remains a need in the art to optimize the usefulness of pro-apoptotic peptides, for example, for use in approaches to cancer chemotherapy. In particular, it would be useful to be able to identify potential apoptotic peptides based on features such as their sequence and physical characteristics. Apoptotic peptides could then be used to induce apoptosis in target cells of interest.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods of identifying potential apoptotic peptides (PAPs). In particular, invention methods are based on modelling the properties of the antibacterial peptides that present selectivity for bacteria (and thus have very low toxic effects on mammaUan cells when apphed extracellular ly). Invention methods provide a sequence-pattern recognition approach to detect peptides that will be toxic towards mitochondria but not to mammaUan ceUs when appUed extracellularly. Apoptotic peptides identified by invention methods induce apoptosis by sweUing mitochondria when targeted intracellularly, as previously described (Ellerby et al, supra). Invention methods are collectively referred to as APAP, as an abbreviation for Approach for detecting PAPs.

Using APAP of the present invention, substance P (SP), an extensively studied neuropeptide present in mammals, birds and fish, was found to have all the sequence characteristics of the PAPs. According to further invention methods, it was shown that SP is capable of swelling mitochondria and inducing the cleavage of caspase-3 zymogen, a known substrate of the active form of caspase-9 in vitro. SP demonstrated very low toxicity for eukaryotic ceUs when appUed extraceUularly, in addition to displaying toxicity towards bacterial ceUs. Thus, invention methods were used to identify a new PAP using a sequence-pattern recognition approach, and suggest a new role for SP in the brain.

In accordance with one aspect of the present invention, there are provided methods of identifying PAPs comprising determining the likelihood of heUcity and the isoelectric point of a given peptide sequence, and comparing these values to those of known antibacterial peptides, apoptotic peptides, or peptides with known high therapeutic indices. In a preferred embodiment, the UkeUhood of heUcity is determined under more than one set of conditions, such as in aqueous solution and in the presence of a charged membrane. In specific embodiments, calculations are made using the AGADIR score and the heUcal hydrophobic moment (M) of the given peptide sequence.

In accordance with another aspect of the present invention, there are provided methods of inducing apoptosis in a target cell using peptides identified by the previously described invention methods. In preferred embodiments, the target cell is a cancer cell. In specific embodiments, the peptide is one identified in Table 3, such as substance P.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the pro-apoptotic activity of substance P (SP). The release of cytochrome c from mitochondria and the processing of caspase-3 into the active form are shown for SP and controls (sonication, the detergent Triton X-100 and a non-toxic peptide DLSLARLATARLAI; SEQ ID NO:l).

Figure 2 coUectively shows the selective toxicity of SP on bacteria. The effect of

SP and the C-terminal caspase cleavage peptide fragment C31 (known to be apoptotic) was determined. Figure 2a shows the effects on ceU viability on fibroblast ceUs. Figure 2b shows the effects on bacteria ceUs. The viabiUty is reported relative to a control peptide (DLSLARLATARLAI; SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the present invention, there are provided methods of identifying PAPs comprising determining the UkeUhood of heUcity and the isoelectric point (IP) of a given peptide sequence, and comparing said likelihood of heUcity and said IP to the UkeUhood of heUcity and IP of a plurahty of known antibacterial peptides to identify a PAP. In a preferred embodiment, the UkeUhood of heUcity is determined under more than one set of conditions, such as in aqueous solution and in the presence of a charged membrane, more preferably a negatively charged membrane. In specific embodiments, calculations are made using the AGADIR score and the heUcal hydrophobic moment (M) of the given peptide sequence.

In order to optimize the pro-apoptotic approach to target and kill angiogenic endotheUal ceUs supplying cancer cells, invention methods provide APAP, an approach to detect PAPs. APAP was originaUy developed to overcome the problems of toxicity and synthesis associated with chemotherapeutic approaches (EUerby et al, supra). Positively charged PAPs, which are non-toxic outside the ceU, are targeted to rumor vasculature by fusion with a peptide that recognizes a receptor on the ceU surface (Pasqualini et al, Nat. Biotechnol. 15:542-6, 1997; Hartetal, /. Biol. Chem. 269:12468-74, 1994); and consequently internalized where they disrupt the negatively charged mitochondria, thereby exerting their pro-apoptotic effect.

The amphipathicity, isoelectric point (IP) and AGADIR scores for a subset of 30 different antibacterial peptides was calculated. The values grouping those peptides with the highest therapeutic index (TI) were considered the signature of PAPs. PAPs resembling antibacterial ones were screened, based on the posited relationship between mitochondrial-dependent apoptosis mechanisms and antibacterial activity. Thus, invention methods provide a tool to identify PAPs independently of any sequence similarity with other known antibacterial or pro-apoptotic peptides. Additionally, invention methods aUow one to search sequence databases systematicaUy.

The amphipathicity and IP were calculated because amphipathic peptides are known to be membrane-associated (Eisenberg et al, T.C. supra), and the selectivity for recognizing bacterial-like membranes depends on the composition of the membranes (Oren and Shai, supra; Matsuzaki et al, supra). AdditionaUy, it has been previously recognized that hydrophobic peptides display both antibacterial activity and toxicity against mammaUan ceUs (Kiyota et al, Biochemistry 35:13196-204, 1996) (i.e., non-selective toxicity), thus PAPs would be expected not to be simply highly hydrophobic peptides. Table 1 shows the peptide sequences of a subset of antibacterial peptides. AU of the peptides but one in Table 1 were hydrophilic, constituting an appropriate group of peptides from which to select PAPs. It has been shown previously that antibacterial peptides with lower hydrophobicity display higher specificity towards gram-negative bacteria (Dathe et al, FEBS Lett. 403:208-12, 1997). In agreement with this notion, aU the peptides analyzed herein presented higher specificity towards Gram negative (G(-)) bacteria as expressed by the TI values (see Table 2).

Table 1. Peptide sequences of a subset of antibacterial peptides.

Alternatively, the propensity to form soluble structures in water (expressed by the propensity to form secondary structures in water, AGADIR score) was used. Since hydrophobicity and the propensity to form soluble structures in water are inversely related, it is expected that hydrophobic sequences will display a low AGADIR score. The inverse is not necessarily true, though; that is, peptide sequences with low AGADIR scores are not necessarily hydrophobic. Interestingly, PAPs tend to be hydrophiUc with low AGADIR scores (Table 2B and Table 3).

Table 2A. Observed characteristics of a subset of antibacterial peptides.

Peptide: See Table 1 for the amino acid composition for each peptide described in this table.

CD Observed in water or Upid: % of alpha-heUcal secondary structure determined by circular dichroism.

Antibacterial activity G(+) or G(-): the minimal inhibitory concentration (μg/mL) for each peptide against Gram positive G(+) and Gram negative G(-) bacterial ceUs.

Cytotoxicity: the concentration (μg/mL) required for inhibiting the growth of mammaUan ceUs, usuaUy red blood cells or fibroblasts. Table 2B. Calculated characteristics of a subset of antibacterial peptides.

A: AGADIR score

M: Average heUcal hydrophobic moment

IP: Estimated isoelectric point

<H> Averaged hydrophobicity

TI: Calculated therapeutic index. The therapeutic index (TI) of a peptide, as used herein, refers to the ratio between the inhibitory concentration observed with mammalian cells and the inhibitory concentration observed with bacterial ceUs. The higher the value of this ratio, the more specific the peptide is for prokaryotic (negatively charged) membranes. A peptide is considered herein to have a high therapeutic index is TI is > 2.5, preferably >5.0, more preferably >10, most preferably >100.

The "signature" of a peptide as used herein, refers to the profile of values obtained from UkeUhood of heUcity parameters (both AGADIR (A) and M values), and optionaUy combining into this profile the calculated IP of the peptide. For example, the signature of the peptide cecropin A in Table 2B would be A=1.2, M=0.44, IP=11.2. In comparing the UkeUhood of heUcity and IP of an unknown peptide to known antibacterial peptides, known apoptotic peptides, or peptides with known high therapeutic indices, the following values are indicative (i.e., typical signatures) of potential apoptotic peptides (PAPs): (i) A <10, preferably <5; (ii) M in the range of about 0.4 - about 0.6; and (in) IP in the range of about 10-13, preferably 10.5 - 12.0, most preferably 10.8 - 11.7. In comparing values, the AGADIR and M values, and optionaUy the IP value are compared between unknown and known peptides; wherein values in the above ranges are indicative of a PAP. Thus, peptides that have similar signatures, would have at least A and M in the above ranges; and optionally A, M and IP in the above ranges.

Table 3. Potential Apoptotic Peptides in the SwissProt database.

SwissProt name: The accession name in the SwissProt database for that particular peptide.

A: AGADIR score

M: Average heUcal hydrophobic moment IP: Calculated isoelectric point <H> Averaged hydrophobicity

Cyto=Cytotoxicity: the concentration (μg/mL) required for inhibiting the growth of mammalian cells, usuaUy red blood ceUs or fibroblasts.

The peptides used to define the parameters of the PAPs (Table 1) are mostly synthetic peptides, with the exception of three natural peptides (rnagainin, cecropin A and meUttin). None of these three natural peptides in Table 1 were detected in the analysis because they were deposited in the SwissProt database in their mature form. In this form, they were longer than the cut-off value used to define the peptide database analyzed. Alternatively, two Cecropins (cec4_bommo, cecb_antpe) and two other natural antibacterial peptides (crbl_vescr, dms3_physa) were found. In agreement with predictions, these antibacterial peptides have been reported to have TIs similar to PAPs (Table 3). As further evidence of the vahdity of invention methods, two peptides, C31 and a control, that did not match the IP, with M and A scores of PAPs were tested for their apoptotic effects (Figure 2). The C31 peptide has been shown to induce apoptosis by an unknown mechanism (Lu et al., Nat. Med. 6:397-404, 2000), so it is considered an interesting target for invention methods, and to provide some hints on the mechanism of action of C31. None of these peptides is toxic to bacterial or mammaUan cells when appUed extraceUularly thus confirming predictions based on invention methods. Based on these results, C31 may induce apoptosis by a different mechanism than PAPs.

In total, 14 sequences were identified as PAPs in the SwissProt database by invention methods. These 14 peptides can be placed into four different groups based on their known function; i.e., antibacterial peptides, neuropeptides, mast cell degranulating peptides and protein-protein interacting peptides. Two out of these four groups, antibacterial peptides and neuropeptides, represent more than 80% of the total (Table 3). Neuropeptides appear to be over-represented since there were only 48 neuropeptides in the original pool of 2,473 peptides in the SwissProt database. The special need for antibacterial peptides in the mammaUan brain has been pointed out previously (Boman, Ann. Rev. Immunol. 13:61-92, 1995), since these may represent a more immediate line of control for bacterial infection than the immune system (which has restricted access to the brain). Considering the properties of PAPs, the present invention suggests that some previously identified neuropeptides may have antibacterial activity.

Among the neuropeptides identified as PAPs (Table 3), 4 were homologues of SP: tkna_gadmo, tkna_horse, tkna_oncmy, and tkna_scyca. SP belongs to the tachykinin family. Tachykinins are synthesized as larger protein precursors (usuaUy more than 40 amino acids in length) that are enzymaticaUy converted to their mature forms (Maggio, Ann. Rev. Neurosci. 11:13-28, 1988). In the original search, it was possible to detect only those recorded in the SwissProt database in the active form. Analyzing aU of the tachykinins deposited in the database (precursors and active forms), 10 out of 61 were predicted to be PAPs. Notably, these 10 were SP peptides from different species.

SP is known to form an α-helical structure in hydrophobic environments but not in aqueous solution (Keire and Kobayashi, Prot. Sci. 7:2438-50, 1998), while it has a positive charge distribution over its sequence, supporting the finding that SP is a PAP. Therefore, the neuropeptide SP was tested for its preference for mitochondria-Uke membranes. The results support the prediction that SP is a PAP. However, complete inhibition of E. coli growth was not observed, probably because of its weU known short half -life in solution (minutes), while experiments lasted for 8 hours. Another possibiUty is that SP only displays a bacteriostatic activity, since the toxicity displayed by SP on bacterial ceUs was not markedly affected by the concentration of SP, as in the case of antibacterial peptides.

In developing APAP, invention methods focused on the characteristics that define selectivity rather than efficiency to kill bacteria. Therefore, it is not surprising that SP demonstrated bacteriostatic, but not bactericidal, activity. It is noteworthy that SP and most of the antibacterial peptides analyzed in this study (Table 1) are active in the low micromolar concentration range, and that SP is only 11 amino acids long. However, SP was toxic at higher concentrations than the antibacterial peptides in Table 1. Thus, invention methods provide a computational approach, APAP, to identify PAPs. These peptides display selectivity towards bacteria and mitochondria, with Uttle toxic effect on eukaryotic ceUs when appUed extraceUularly, thus providing the basis for a new generation of drugs that can be present in the body without toxic effect unless they are taken in by targeted ceUs (EUerby et al, supra). From a pubUc database, the approach detected mostly antibacterial peptides and neuropeptides suggesting that these neuropeptides may be the first reported with antibacterial activity. In agreement with this idea, SP is a PAP with a TI > 100. These activities have likely been present in SP during the course of evolution of the tachykinins, which would support the possibiUty of a biological significance for these findings. APAP provides a method to detect and ultimately improve pro-apoptotic peptides for chemotherapy.

The invention will now be described in greater detail by reference to the following non-limiting examples.

EXAMPLE 1 Sequence-Pattern Recognition Approach

Known antibiotic peptides were found to fit a pattern, which includes a low UkeUhood of heUcity in aqueous solution, a high likelihood of heUcity in the presence of negatively charged membranes, and a high isoelectric point (IP). The heUcal probabiUty of monomeric peptides in aqueous solution (AGADIR score), the IP and the hydrophobic moment were calculated to account for the characteristics of antibacterial peptides with low toxic activity against mammaUan ceUs. These characteristics are important in determining the selectivity observed in these peptides towards bacterial membranes and bacterial-like membranes (i.e., mitochondrial membranes).

A subset of 30 antibacterial peptides previously reported in the Uterature was used for calculations of AGADIR scores (A) (Munoz and Serrano, Nat. Struct. Biol 1:399- 409, 1994), IP, and average heUcal hydrophobic moments (M) (Eisenberg et al, supra), (Table 2). The peptide sequences of this subset are shown in Table 1. The TI of a peptide is here defined as the ratio between the inhibitory concentration observed with mammaUan ceUs and the inhibitory concentration observed with bacterial ceUs (Table 2). The higher the value of this ratio is, the more specific the peptide is for prokaryotic (negatively charged) membranes.

PAPs were searched for in the SwissProt database, release 38 (Bairoch and Apweiler, Nucl. Acids Res. 27:49-54, 1999), which contains a total of 80,000 protein sequences. First, aU of the peptide sequences of 40 or fewer amino acids in length were extracted from this database. Then aU of these sequences (2,473 database entries) were used to calculate their corresponding M, IP and AGADIR scores. Protein fragments, as opposed to peptides, were not considered herein.

In order to reproduce these biophysical properties, three scores from the sequences of these peptides were calculated. Table 2A shows a subset of selected antibacterial peptide sequences and the corresponding experimental values for helix formation in water and in hydrophobic environments, antibacterial activity and cytotoxic activities against mammaUan ceUs. Table 2B shows the corresponding calculated values for M, IP, A and the TI. The antibacterial peptides presented in Table 1 are more potent against G(-) (MIC=17.3 μg/mL on average) bacteria than to G(+) (MIC=44.3 μg/mL), and we used the G(-) values as a reference for the TI.

Peptide sequences with values ranging from 0.4<M<0.6, A<10.0 and 10.8<IP<11.7, were found to have the highest TI (highest specificity for bacteria) (Table 2B). These parameters were therefore hypothesized to be the signature of the PAPs. Searching for PAPs in the SwissProt Database led us to identify 14 PAPs (Table 3). Two of these peptides have previously been characterized with respect to their toxicity against bacteria and mammalian ceUs, and in both cases a greater toxicity towards bacterial ceUs was observed (Table 3).

EXAMPLE 2 Computational Resources

The PEPPLOT and ISOELECTRIC programs from the GCG package (Wisconsin package version 10, USA) were used to calculate M and IP, respectively. The non-zero alpha values calculated by the PEPPLOT program were averaged for windows of eight residues. To calculate the AGADIR score, the AGADIR program, which was kindly provided by Dr. Luis Serrano at EMBL, was used. The hydrophobicity of peptide sequences was obtained by calculating the average hydrophobicity of the sequence using the consensus scale reported by Eisenberg (Eisenberg et al, supra). AU these programs were run on an SGI Origin 2000 server.

EXAMPLE 3

Caspase-3 Activation in a Cell-Free Apoptosis System Induced by SP.

Cytoplasmic extracts were prepared as described before (Hart et al, supra). Briefly, non-apoptotic neuronal cells were sonicated and centrifugated at 16,000 g. This extract was made free of nuclei, mitochondria and did not self -prime.

Rat and mouse Uver mitochondria were prepared as described by Hovius et al.

(Hovius et al, Biochim. Biophys. Ada 1021:217-26, 1990), with modifications as described previously (EUerby et al, J. Neurosci. 17:6165-78, 1997). Cultured ceU mitochondria were prepared as described previously (Moreadith and Fiskum, Anal. Biochem. 137:360-7, 1984).

Electrophoresis of proteins was carried out using either 8% or 12% SDS- polyacrylamide gels. Equal amounts of total protein were loaded per lane, and the proteins were separated at 4 °C at 50 V through the stacking gel, and 90 V through the separating gel.

Western blot transfer of the proteins separated by electrophoresis was carried out at 4 °C using PVDF Membranes (0.2 mm) (Biorad), at either 200 mA for two hours. Blots were then blocked for 1 hour in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween) containing 5% non-fat dried milk. FinaUy, the membranes were probed with an appropriate dilution (1:500 to 1:2000) of primary antibody in TBST containing 5% non-fat dried milk for 1-4 hours, depending upon the antibody.

Anti-caspase-3 antibodies from mouse, rabbit and goat were purchased from

Transduction Laboratories, Inc., Upstate Biotechnology, Inc. and Santa Cruz Biotechnology, Inc., respectively. The blots were washed three times for 1 hour with TBST, f oUowed by incubation in a peroxidase-coupled secondary antibody for 1 hour in TBST containing 5% non-fat dried milk. The mouse, human, and rabbit peroxidase-coupled secondary antibodies were from Amersham. Enhanced chemiluminescence detection of the proteins was carried out using Hyperfilm ECL (Amersham), and with Pierce SuperSignal Substrate Western Blotting reagents, or Amersham ECL reagents.

EXAMPLE 4 Mitochondrial Swelling Assays

Rat Uver mitochondria were prepared as described above. The peptide concentrations used to sweU mitochondria were 50 μM L-Substance P, 10 μM D-

(LSLARLATARLAI; SEQ ID NO:34) (negative control), or 200 μM Ca⁺² (positive control). The swelling was quantified by measuring the optical absorbance at 540 nm.

One of the PAPs identified, SP, was tested for its abiUty to swell mitochondria and induce caspase-3 activation in a ceU-free system. This system was developed previously to simulate neuronal apoptosis (EUerby et al, 1997, supra). SP induces the swelling of mitochondria at 50 μM. At such concentration, SP was capable of releasing cytochrome c from mitochondria and activating caspase-3 (Fig. 1). In contrast, a peptide chosen as negative control which did not present the properties of PAPs did not display any observable effect on mitochondria (Fig. 1).

EXAMPLE 5

Activity of SP

10⁴ human embryonic kidney 293 ceUs per weU were seeded into a 96 weU plate. After 20 hours, different aqueous dilutions (Fig. 2B) of SP (SIGMA, USA), C31 and a peptide used as a control were added to the culture and the ceU death was quantified by try pan blue exclusion 48 hours later.

Toxicity of SP for Bacterial CeUs.

DH5α E. coli ceUs were grown overnight as a pre-inoculum for the bacterial culture used in this assay. When the ceUs were at the end of their log phase (optical density at 600 run of 0.8-1.0), 1 μL was used to inoculate 5 mL. Such dilution produced initial concentrations of bacteria capable of forming 10⁵-10⁶ colonies per mL in LB plates at 37 °C, that is 10⁵-10⁶ colony forming units. AU the bacterial cultures used in these experiments were grown in LB at 37 °C. The concentration of SP required to inhibit the ceU growth by 60% was determined by foUowing bacterial growth in LB Uquid in the presence of varying concentration of the peptide: 0, 1, 10, 20, 50, 125, and 250 μM. Sterilized 96-weU plates of polystyrene with flat bottom and low evaporation lid (Costar, USA) were used, in a final volume of 100 μL: 50 μL of LB containing 10⁵-10⁶ colony forming units, and 50 μL of LB with a two-fold dilutions of the peptide. A 10 mM stock solution of the peptide was prepared with 5 mg of SP in 371 μL of water. Inhibition of growth was detected by measuring optical density at 600 run with a microplate spectrophotometer SPECTRAmax (Molecular Devices, USA) at varying times: 0, 3, 5, 6, 7 and 8 hours. Each ICβo was determined from at least two independent experiments performed in tripUcate. AdditionaUy, the colonies formed from each experiment were counted in LB plates at 0 and 8 hours of growth.

The toxicity of SP against bacteria was tested and compared to the effect of SP on fibroblasts when appUed extraceUularly. SP was able to reduce the growth of E. coli cells with an ICβo of 10 μM (Fig. 2B). By comparison, the negative control peptide did not have any toxicity against bacteria. In contrast, Fig. 2A shows that SP did not affect the growth of fibroblasts when appUed extraceUularly even at a concentration of 1 mM. These results indicate that SP has a TI > 100. Additionally, a peptide from the protein APP (the last 31 amino acids in APP, referred as C31) known to induce apoptosis when expressed intraceUularly (Lu et al, supra) was tested for its toxicity against bacteria and mammaUan ceUs. This peptide did not present the properties (IP, M, A scores) of PAPs. C31 did not present any observable toxicity against bacterial or mammaUan ceUs when appUed extraceUularly (Fig. 2 A and 2B).

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed. The contents of the articles, patents, and patent appUcations, and aU other documents and electronicaUy available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual pubUcation was specificaUy and individuaUy indicated to be incorporated by reference. AppUcants reserve the right to physicaUy incorporate into this appUcation any and aU materials and information from any such articles, patents, patent appUcations, or other documents.

Claims

That which is claimed is:

1. A method of identifying a potential apoptotic peptide (PAP), said method comprising: determining the UkeUhood of heUcity and the isoelectric point (IP) of a given peptide sequence to obtain a signature for said peptide sequence; comparing said signature to the signatures of a pluraUty of known antibacterial peptides; and identifying peptides that have a signature similar to the signatures of said pluraUty to be PAPs.

2. A method according to claim 1, wherein said determining comprises calculating the UkeUhood of heUcity of said peptide sequence under more than one set of conditions.

3. A method according to claim 2, wherein said UkeUhood of heUcity is calculated in aqueous solution and in the presence of a charged membrane.

4. A method according to claim 3, wherein the heUcity in aqueous solution is calculated by the AGADIR score of the peptide sequence.

5. A method according to claim 3, wherein the heUcity in the presence of a charged membrane is calculated by the heUcal hydrophobic moment (M) of the peptide sequence.

6. A method according to claim 3, wherein the heUcity in aqueous solution is calculated by the AGADIR score of the peptide sequence and the heUcity in the presence of a charged membrane is calculated by the heUcal hydrophobic moment (M) of the peptide sequence.

7. A method according to claim 6, wherein an AGADIR score of less than 10 and a M value of about 0.4 to about 0.6 is indicative of a PAP.

8. A method according to claim 7, wherein an IP of about 10 to about 13 is further indicative of a PAP.

9. A method of identifying a potential apoptotic peptide (PAP), said method comprising: determining the likelihood of heUcity and the isoelectric point (IP) of a given peptide sequence to obtain a signature for said peptide sequence; comparing said signature to the signatures of a pluraUty of known apoptotic peptides; and identifying peptides that have a signature similar to the signatures of said pluraUty to be PAPs.

10. A method of identifying a potential apoptotic peptide (PAP), said method comprising: determining the UkeUhood of heUcity and the isoelectric point (IP) of a given peptide sequence to obtain a signature for said peptide sequence; comparing said signature to the signatures of a pluraUty of peptides, wherein each of said plurality of peptides has a known high therapeutic index; and identifying peptides that have a signature similar to the signatures of said plurality to be PAPs.

11. A method of inducing apoptosis in a target ceU, said method comprising contacting a target ceU with an effective amount of a peptide identified according to claim 1.

12. A method according to claim 11, wherein said target ceU is a cancer ceU.

13. A method according to claim 11, wherein said peptide is selected from the group consisting of the potential apoptotic peptides Usted in Table 3.

14. A method of inducing apoptosis in a target ceU, said method comprising contacting a target ceU with an effective amount of substance P.

15. A method of inducing apoptosis in a target ceU, said method comprising contacting a target ceU with an effective amount of a peptide identified according to claim 9.

16. A method of inducing apoptosis in a target ceU, said method comprising contacting a target ceU with an effective amount of a peptide identified according to claim 10.