GB2628421A

GB2628421A - Peptides and uses thereof

Info

Publication number: GB2628421A
Application number: GB2304356.5A
Authority: GB
Inventors: Warenius Hilmar
Original assignee: Syntherix Ltd
Current assignee: Syntherix Ltd
Priority date: 2023-03-24
Filing date: 2023-03-24
Publication date: 2024-09-25
Also published as: GB202304356D0; WO2024200264A1

Abstract

A cyclic peptide comprising an active region and a cassette region, wherein the active region comprises the amino acid sequence X1X2X3X4X5X6 and a cassette region which comprises the amino acid sequence Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg- Arg, or a salt thereof, wherein: X2 and X5 are arginine; and wherein: X1, X3, X4 and X6 are any non-polar amino acid selected from (7-methoxy-coumarin- 4-yl)-Ala-OH (Dac), sarcosine (Sar), 3-amino-3-(2-napthyl) propionic acid (Nap), 5,5- dimethylproline (dmPro), and 3-(2-Naphthyl)-alanine (Nal). The exemplified peptide may comprise the sequence: Dac-Arg-Sar-Nal-Arg-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg. The invention further relates to a pharmaceutical composition comprising the cyclic peptide and the peptide for use in medicine, and particularly for the treatment of cancer.

Description

PEPTIDES AND USES THEREOF

Field of the invention

The present disclosure relates to cyclic peptides and medical uses of such peptides, particularly for the treatment of cancer.

Background

The somatic mutation theory has been the prevailing paradigm in cancer research for several decades According to this theory, cancer is derived from a single somatic cell that has accumulated multiple DNA mutations in proto-oncogenes and tumour suppressor genes which results in unconstrained cell division and tumour formation.

In addition to somatic mutations which drive oncogenesis, cancer cells typically express a range of disparate characteristics including the avoidance of apoptosis, reliance on aerobic glycolysis and CDK4 overexpression.

Normal cells either repair mutational DNA damage or undergo the energy-dependent process of apoptosis. Cancer cells typically evade apoptosis, allowing accumulation of further mutations and dysregulated growth. This may he achieved by increasing or decreasing the expression of anti-or pro-apoptotic genes, respectively, or by stabilizing or dc-stabilizing anti-or pro-apoptotic proteins, respectively. Consequently, the inhibition of anti-apoptotic proteins and induction of apoptosis in cancer cells has become an important focus for the development of cancer therapeutics.

Apoptosis is an energy-dependent process. The relative availability of ATP is a major factor in determining whether or not apoptosis can occur in a normal cell in response to a mutation, whether oncogenic or non-oncogenic. High levels of ATP enable cells to undergo apoptosis, and low levels of ATP shift cells away from apoptosis towards necrosis. Cancer cells navigate a narrow path between apoptosis and necrosis based on their ATP levels. Spontaneous cancer cell necrosis has been observed in many established human cancer cell lines, even under optimal growth conditions.

The availability of ATP in cancer cells, and consequently, the ability to undergo apoptosis, is primarily governed by carbohydrate metabolism. It is known that carbohydrate metabolism in cancer cells, in contrast to normal cells, is restricted to the glycolytic pathway, avoiding the much higher energy-producing mitochondria' respiration. This phenomenon has been termed "aerobic glycolysis". Aerobic glycolysis is the process by which glucose is oxidised to pyruvatc, and subsequently converted to lactate, under normoxic conditions. Aerobic glycolysis is distinct from its counterpart, anaerobic glycolysis, which occurs under hypoxic conditions. The reduced level of ATP produced as a result of aerobic glycolysis and avoidance of mitochondria] respiration may enable cancer cells to avoid apoptosis. Furthermore, aerobic glycolysis facilitates an increased rate of glucose hydrolysis, enabling cancer cells to successfully compete with normal cells for glucose uptake to maintain uninterrupted growth.

Turning to CDK4, CDK4 is a critical mediator of cellular transition from the G1 to the S phase, and its deregulation or overexpression favours the growth and survival of several cancer types. On forming a complex with cyclin D, CDK4 acts classically as a kinase phosphorylating the Rb protein and releasing the transcription factor E2F. E2F promotes expression of target genes which are responsible for the progression through the G1 phase. Consequently, CDK4 inhibitors have been implicated as possible therapies for several cancer types.

CDK4 knockout mice with normal cyclin D activity are resistant. to the induction of cancer by chemical carcinogens (despite the continuing presence of CDK2 and CDK6) confirming the importance of CDK4 in the pathogenesis of cancer. However, other studies have demonstrated that overexpression of CDK4 in human A2780 ovarian cancer cells is accompanied by a corresponding rise in CDK1 expression, but surprisingly, without a corresponding increase in RB phosphorylation. This indicates the possibility of a non-kinase activity of CDK4 in addition to its classical kinase role when complexed with cyclin Dl.

Sequence analysis of the C-terminal regions of CDK4, CDK2 and CDK6 have enabled the identification of a unique decapeptide region in CDK4, between amino acid residues 249 and 258, which has no similar region in CDK6 or CDK2. This has been termed the non-kinase (NK) region of CDK4. The central portion of the NK region comprises the hexapeptide sequence Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP).

The PRGPRP (SEQ ID NO: I) peptide has been artificially synthesised by automated methods. When tested in tissue culture on MGHU1 bladder cancer cells the hexapeptide was found to be cytotoxic, albeit requiring a high (milli molar) concentration, and a protracted period of exposure.

Peptides variants which arc based on the NK region of CDK4 have been described in WO 2009/112536. Specifically disclosed are cyclic peptides composed of an PRGPRP "warhead" and an amphiphilic "backbone" forming a 16-18 amino acid cyclic peptide. One such peptide, El I L R-001/TH R53 (Pro-A rg-Gl y-Pro-A rg-Pro-V al-Al a-Leu-Ly s -Leu-Al a-Leu-Ly s -Leu-Al aLeu) (SEQ ID NO:2), exhibited selective killing of H460 human non-small cell lung cancer cell lines by necrosis without affecting primary diploid human fibroblasts, keratinocytes or MRCS cells, in vitro. The necrosis was accompanied by a fall in intracellular ATP levels. However, although such peptides had anti-cancer effects, they were not candidates for in vivo cancer therapy due to high concentrations being required to obtain a local effect.

Attempts have been made to improve the efficacy of cyclic peptides comprising the "PRGPRP" warhead, by increasing cell membrane penetration. HILR-025 (Pro-Arg-Gly-Pro-Ara-Pro-ValTrp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Tip-Trp) (SEQ ID NO:3), as described in WO 2016/020437, showed a significant improvement in cancer cell killing in vitro without lethality in MRC-5 cells. However, the specific activity of HILR-025 was not sufficiently high for further development as an in vivo therapeutic. In addition, some studies indicated that the increased anti-cancer effect was, in part, due to a non-specific contribution to cell killing by the HILR -25 backbone.

Additional anti-cancer peptides which are analogues of the "PRGPRP" warhead have been described in WO 2020/193978. Specifically disclosed are cyclic peptide sequences comprising Dac-Arg-Sar-Nap-Arg-Nap (SEQ ID NO:4) or Dac-Arg-Sar-dmPro-Arg-Nap (SEQ ID NO:5) (where Dac is 7-methoxy-coumarin-4-yl)-Ala-OH, Sar is sarcosine, Nap is 3-amino-3-(2-naphthyl) propionic acid and dmPro is 5,5-di methylprol ine) which show a further improvement in cancer cell killing in vitro.

There remains the need to identify new compounds which have improved cytotoxic effects against cancer cells.

Summary

Accordingly, in a first aspect, the present invention provides a cyclic peptide comprising an active region and a cassette region, wherein the active region comprises the amino acid sequence X1X2X3X4X5X6 and a cassette region which comprises the amino acid sequence ValTrp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO:6), or a salt thereof, wherein: X= and X' are argininc; and wherein: X', X3, X4 and X6 are any non-polar amino acid selected from (7-methoxy-coumarin-4-y1)-Ala-OH (Dac), sarcosine (Sar), 3-amino-3-(2-napthyl)propionic acid (Nap), 5,5-di methylproline (dmPro), and 3-(2-Naphthyp-alanine (Nal).

In a second aspect, the present invention provides a pharmaceutical composition comprising a cyclic peptide as described above, and a pharmaceutical acceptable carrier, diluent or excipient.

In a third aspect, the present invention provides a cyclic peptide as described above, for use in medicine.

Preferred features are set out in the appended claims.

It has surprisingly been found that the cyclic peptides according to the present invention may be used to achieve selective killing of cancer cells at a low concentrations of the peptides. A low concentration of the peptides prevents unwanted side-effects often associated with traditional cancer treatments such as chemotherapy.

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Nor is the claimed subject matter limited to implementations that solve any or all of the disadvantages noted herein.

Brief Description of the Drawings

To assist understanding of the present disclosure and to show how embodiments may be put into effect, reference is made, by way of example only, to the accompanying drawings in which: Figure 1 is a graph illustrating human non-small cell lung cancer cell survival following treatment with cyclic PRGPRP peptide (SEQ ID NO:1).

Figure 2 is a graph illustrating human non-small cell lung cancer cell survival following treatment with H I LR-025 (cyclic Pro-Arg-Gly-Pro-Arg-Pro-Val-Trp-Trp-Arg-Arg-Trp-TrpArg-Arg-Trp-Tip) (SEQ ID NO:3).

Figure 3 is a graph illustrating human non-small cell lung cancer cell survival following treatment with H I LR-055 (cyclic Cys-Dac-Arg-Sar-Nal-Arg-Nal-Cys) (SEQ ID NO: 7) Figure 4 is a graph illustrating human non-small cell lung cancer cell survival following treatment with HILR-17 (cyclic Cys-Dac-Arg-Sar-Nap-Arg-Nap-Cys) (SEQ ID NO:8) Figure 5 is a graph illustrating human non-small cell lung cancer cell survival following treatment with HILR-56 according to an example of the invention (cyclic Dac-Arg-Sar-NalArg-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ 1D NO: 9)

Detailed Description

Through detailed investigations, the present inventor has surprisingly found that the introduction of specific highly non-polar amino acid residues in the previously disclosed ProArg-Gly-Pro-Arg-Pro (SEQ ID NO:1) peptides, and further combination with an amphiphilic cassette, results in a significantly improved cytotoxicity against cancer cells.

Accordingly, as indicated above, in a first aspect, the present. disclosure is concerned with a cyclic peptide comprising an active region and a cassette region, wherein the active region comprises the amino acid sequence X'X2X3X4X5X6 and the cassette region comprises the amino acid sequence Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO:6), or a salt (hereof, wherein: X= and X5 are arginine; and wherein: X', X3, X' and X6 are any non-polar amino acid selected from (7-methoxy-coumarin-4-yI)-Ala-OH (Dac), sarcosine (Sar), 3-amino-3-(2-napthyl)propionic acid (Nap), 5,5-dimethylproline (dmPro), and 3-(2-Naphthyl)-alanine (Nal).

The terms used in this specification generally have their ordinary meanings in the art, within the context of the present disclosure, and in the specific context where each term is used. Certain terms that are used are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the present methods and devices.

The term "comprising" or "comprises" as used herein denotes the inclusion of at least the features following the term and does not exclude the inclusion of other features which have not been explicitly mentioned. The term may also denote an entity which consists of features following the term.

Various embodiments are described in detail and may be further illustrated by the provided Figures. As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

The term "amino acid" as used throughout the specification is not limited to only naturally occurring amino acids but also includes non-standard unnatural amino acid residues. As such, at least some of the sequences described throughout the specification comprise non-standard unnatural amino acid residues. Throughout the present disclosure, the abbreviation "Sar" refers to the amino acid residue sarcosine. "Dac" refers to the amino acid residue (7-methoxycoumari n-4-y1)-Al a-OH. "Dm-Pro" refers to the amino acid residue 5, 5-di methylprol inc. "Nap" refers to the amino acid residue 3-amino-3-(2-naphthyl)propionic acid. "Nat" refers to the amino acid residue 3-(2-Naphthyl)-alaninc. Unless otherwise specified, the stereoisomeric form of the amino acids mentioned herein is not limited. As such, the stereoisomeric form of the amino acids may be the L-cnantiomcr or the D-cnantiomcr. hi other examples, the amino acids may be in the (R) or (5) configuration.

Active Region In sonic embodiments, X1 is Dac. In other embodiments, X3 is Sar. In further embodiments, X4 and X6 are Nal. Other permutations and combinations of these amino acids arc envisaged such that in some embodiments, X' is Dac and/or X3 is Sar and/or X4 and X6 arc Nal. The active region of the cyclic peptide may comprise any of the following sequences: X' is Dac, X3 may he Sar, and/or X4 and X6 may he Nal; X' is Dac, X3 may be Sar, and/or X4 may be Nap and/or X6 may be Nal; X1 is Dac, X3 may he Sar, and/or X4 may he Nal and/or X6 may he Nap; X1 is Dac, X3 may be Sar, and/or X4 and X6 may be Nap; X1 is Sar, X3 may be Sar, and/or X4 and X(' may be Nal; X' is Sar, X3 may be Sar, and/or X4 may be Nap and/or X6 may be Nal; X1 is Sar, X3 may be Sar, and/or X4 may he Nal and/or X6 may be Nap; X' is Sar, X3 may be Sar, and/or X4 and X6 may be Nap; X' is Dac, X3 may be Dac, and/or X4 and X6 may he Nal; X' is Dac, X3 may be Dac, and/or X4 may be Nap and/or X6 may be Nal; X' is Dac, X3 may be Dac, and/or X4 may be Nal and/or X6 may be Nap; X' is Dac, X3 may be Dac, and/or X4 and/or X6 may be Nap; X' is Sat-, X3 may be Dac, and/or X4 and/or X6 may be Nal; X' is Sar, X3 may be Dac, and/or X4 may be Nap and/or X6 may be Nal; X1 is Sar, X3 may he Dac, and/or X4 may he Nal and/or X6 may he Nap; X1 is Sar, X3 may be Dac, and/or X4 and X6 may be Nap.

Preferably, at least Nal is provided as the L-enantiomer (i.e. 3-(2-Naphthyl)-L-alanine) in the peptides of the invention. The inventor has found that when Nal is provided as a D-enantiomcr (i.e.as 3-(2-Naphthyl)-D-alanine), the cytotoxicity effects of the corresponding active region are diminished. More preferably, all the amino acids of the active region and/or cassette region are provided as their respective L-enantiomers. In other embodiments, the amino acids of the active region and/or cassette region are a mixture of L-and D-enantiomers. (Sarcosine lacks a chiral centre and exists in one form only.) The present inventor has also surprisingly found that the length of the active region of the peptide has an effect on the cytotoxic activity of the peptides. Without being bound by theory, it is believed that conformational constraint of the peptides is likely to play a part in their activity.

In some embodiments of the invention, the active region of the peptide is between 6 and 10 amino acids in length. In these embodiments of the invention, the active region of the peptide may he 6, 8 or 10 amino acids in length. Preferably, the active region of the peptide is 6 amino acids in length. The present inventor has surprisingly shown that a peptide length of 6 amino acids results in a higher level of cancer cell cytotoxicity.

In preferred embodiments, the active region of the cyclic peptide comprises the amino acid sequence: Dac-Arg-Sar-Nal-Arg-Nal (SEQ ID NO: 10). Preferably, at least Nal is provided as the L-enantiomer (i.e. 3-(2-Naphthyl)-L-alanine). More preferably, each amino acid of the active region is provided as the L-enantiomer (other than sarcosine which lacks a chiral centre).

In some embodiments, the active region of the cyclic peptide consists of the amino acid sequence: Dac-Arg-Sar-Nal-Arg-Nal (SEQ ID NO: 10) (i.e. the active region of the peptide is 6 amino acids in length.) Cassette Region The cyclic peptides of the present invention comprise an amphiphilic cassette region. The amphiphilic cassette region may enhance the solubility of the active region, and accordingly, facilitates entry of the peptides into cells. The cassette may comprise the following sequence: Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO: 6). In other embodiments, the cassette may comprise an two additional Tip residues and thus comprise the following sequence: ValTrp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp (SEQ ID NO:11) In sonic embodiments, the cassette consists of the following sequences: Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO: 6) or Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp (SEQ ID NO:11). The present inventor has unexpectedly found that removal of the final two Trp residues from the cassette region does not have a significant impact on the cytotoxic activity of the cyclic peptide.

Accordingly, in some embodiments, the present invention provides a cyclic peptide comprising or the following sequence: Dac-Arg-Sar-Nal-Arg-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Ara-Arg (SEQ ID NO:9) In preferred embodiments, the cyclic peptide may consist of the following sequence: Dac-Arg-Sar-Nal-Arg-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO:9) Cyclisation The peptides of the present invention are cyclic. The skilled person will appreciate that there are numerous ways in which the peptides of the present invention could he cycliscd. For example, an amide bond could be formed between a carboxylic acid group and an amine group on the N-and C-termini of the peptide, respectively, with the use of appropriate coupling agents, and optionally. catalysts. Useful coupling agents include, for example, a carbodiimide or uronium derivatives. A particularly preferred coupling agent is EDC Wethyl-3-(3'-dimethykuninopropyl) carbodiimide hydrochloride].

Alternatively, another type of bond or linkage could be formed between the residues at the Nor C-termini of the pcptidc. For example, in some embodiments, the active region of the peptide may be flanked by a cysteine residue at its N-terminus, and the cassette region of the peptide may be flanked by a cysteine residue at its C-terminus. In such embodiments, the peptide may he cyclised by one or more thioester bonds between the flanking cysteine residues.

In other embodiments, the peptide may he cyclised by reacting two thiol groups within the peptide chain with a suitable linker, for example, a di-henzyl bromide.

In preferred embodiments, the active region is contiguous with the cassette region within the cyclic pcptidc.

Other Modifications The peptides of the present invention the peptide may be glycosylated. Cancer cells have altered glucose metabolism, frequently resulting in increased glucose uptake. The increased uptake of '8F labelled Iluorodeoxyglucose in PET clinical scanning for cancer is well-established. Without wishing to be bound by theory, it is therefore expected that such glycosylation increases the uptake of the peptides into cancer cells.

Glycosylation may be achieved by routine methods known to the skilled person. In some embodiments, the peptides may be glycosylated with 2-deoxyglucose at hydroxyl groups which are present in the peptides of the present invention.

Medical Uses In some embodiments, the peptide of the present invention is cytotoxic to, or inhibits the growth of, a cancer cell. In this context, a cancer cell is a cell taken from a primary tumour, a metastasis or other suspected site of cancer in a subject, or a cell line derived from a cancer. It is preferred that the peptide is more cytotoxic to, or inhibitive to the growth of a cancer cell than a noncancerous and/or a control cell. As used herein, the term -non-cancerous cell" is used to mean a normal (e.g. healthy) cell i.e. a cell which does not have a cancerous phenotype. Such cells may be cells from any tissue in a subject. A control cell includes a normal non-cancerous cell and may be derived from the corresponding normal tissue of a patient or from a primary cell culture.

The cyclic peptide of the invention may preferably be combined with one more agents which enhance delivery and/or uptake of the peptide into cells or tissues. Enhanced delivery and/or uptake may be achieved by increasing the solubility of the peptide. Thus, in some embodiments, the agent may comprise a solubility enhancer Agents with any of the above functions would he known to the skilled person. In preferred embodiments, the peptide may be combined with an agent comprising nanoparticles formed of one or more polymers or one or more polymers which are capable of forming nanoparticles. In these embodiments, the polymer may comprise a linear and/or branched or cyclic polymonoguanide/polyguanidine, polybiguanide, analogue or derivative thereof. Such polymers/nanoparticles have been previously described in WO 2020/008195A1 and have been found to enhance the solubility and uptake of the cyclic peptides of the invention. A commercially available agent comprising such nanoparticles is Nanocin-Prom! (Tecrea).

Accordingly, further provided is a formulation comprising a cyclic peptide of the invention and an agent as defined herein. The term "formulation" refers to a mixture, combined preparation and/or composition.

The peptides of the invention described herein are particularly useful in medicine. Thus, in a further aspect of the present invention there are provided medical uses of the peptides of the invention described herein. Specifically provided is a pharmaceutical composition comprising a peptide as described herein, and a pharmaceutical acceptable carrier, diluent or excipient. Accordingly, also provided is the pharmaceutical composition for use in medicine. The skilled person will be familiar with the formulation of pharmaceutical compositions. Any appropriate carrier, diluent or excipient or combinations thereof, may he used. The pharmaceutical composition may alternatively or additionally comprise an agent which enhances delivery and/or uptake of the peptide into cells or tissues as described above. The cyclic peptides or pharmaceutical compositions may specifically be used for the prevention or treatment of cancer.

The peptides of the invention advantageously selectively target cancer cells without having any significant cytotoxic effect on normal, healthy cells because only cancer cells express the PKM2 isotype which causes aerobic glycolysis and is believed to form the target for the peptides. As such, in contrast to conventional treatment regimens for chemotherapy which require periods of rest between pulses of treatment to allow normal cells to recover from the damage caused by chemotherapeutic agents, treatment with the peptides of the present invention may carried out in a continuous manner without periods of rest. For effective treatment, the peptides would need to be maintained at. a critical concentration within cancer cells until necrotic cell death has been achieved, for example, through continuous intravenous infusion. Oral administration or parenteral administration are also envisaged.

The peptides and pharmaceutical compositions of the present invention are effective in treating cancers of various origins, including breast cancer, prostate cancer. colorectal cancer, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer, head and neck cancer, stomach cancer, pancreatic cancer, oesophagus cancer, small cell lung cancer, non-small cell lung cancer, malignant melanomas, multiple myelomas, neuroblastomas, leukaemias, lymphomas, sarcomas and gliomas. Furthermore, the peptides and compositions may be useful for the treatment of a patient suffering from multiple cancer types or metastatic cancer.

The peptides or pharmaceutical compositions may he administered with a further therapeutic agent, for example anti-cancer hormones, chemotherapeutic drugs and/or ionising radiation. In some embodiments, the peptides or pharmaceutical compositions may be administered as part of a treatment regime with one or more conventional therapies such as chemotherapy, radiation therapy or surgery.

Further Aspect In a further aspect, there is provided a cyclic peptide comprising an active region and a cassette region, wherein the active region comprises the amino acid sequence X' X2X3X4X5X6 and a cassette region which comprises the amino acid sequence Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO:12), or a salt thereof, wherein: X2 and X5 are arginine; and wherein: XI, X3, X4 and X6 are any non-polar amino acid selected from (7-methoxy-coumarin-4-yI)-Ala-OH (Dac), sarcosine (Sar), 3-amino-3-(2-napthyl) propionic acid (Nap), 5,5-di methylproline (dmPro), and 3-(2-Naphthyp-alanine (Nal).

In this aspect, the cassette region, cyclisation, modifications, and other features of the peptide and its uses may be as defined herein. The cyclic peptide may also be provided as a formulation or pharmaceutical composition as defined herein.

Cyclic peptides according to this aspect exhibit improved cytotoxicity against cancer cells as compared to known anti-cancer peptides mentioned herein.

Mechanism of Action As mentioned above, an accepted hallmark of cancer cells is their dependency on aerobic glycolysis and avoidance of mitochondria' oxidative phosphorylation.

In the final step of glycolysis, pyruvate kinase catalyses the direct transfer of phosphate from phosphoenolpyruvate to ADP to produce ATP and pyruvate. Current understanding of aerobic glycolysis in the context of cancer is that it arises from the expression of exon 10 rather than exon 9 of the pyruvate kinase gene. Exon 10 expression provides a PKM2 (pyruvate kinase M2) phenotype which is unique to cancer cells and which is responsible for aerobic 12elievel2iz, and exon 9 expression provides a PKM1 (pyruvate kinase M1) phenotype which is found in normal adult cells. PKM I and PKM2 differ in their amino acid sequence between residues 378 and 411. The change in sequence at this location is believed to affect tetramerization of two PK dimers to form an active tetramer responsible for generating ATP from phosphoenolpyruvate.

The PRGPRP amino acid sequence within the NK region in the C-terminal peptide sequence of CDK4 is likely to hind to a unique complementary target sequence (Scr -Asp -Pro -Thr -Glu -Ala (SDPTEA) at residues 406 to 411 of PKM2 in cancer cells. This complementary sequence is located at the site of tetramerization of PKM2 dimers. As tetramerization of PKM2 dimers is required for activation of PKM2, binding of the PRGPRP sequence of the NK region of CDK4 to PKM2 is likely to inhibit ATP production and prevent ATP-dependent apoptosis. In newly transformed cancer cells, an upregulation of CDK4 will, therefore, cause a decrease in ATP levels, thus preventing apoptosis and facilitating unregulated cell growth and division. Accumulation of further mutations in proto-oncogenes and tumour suppression genes may result in full malignant transformation.

It is believed that the peptides of the present invention mimic the NK region of CDK4, and are capable of "hijacking" the mechanism by which cancer cells normally avoid apoptosis. Specifically, by virtue of their high specific activity and binding affinity to the target SDPTEA sequence of PKM2, the peptides have a greater effect than CDK4 on inhibiting 13elievel3ization and activation of PKM2. This results in a more pronounced depletion of intracellular ATP levels compared to that achieved by CKD4 under normal physiological conditions, and results in the killing of cancer cells through necrosis. Moreover, as PKM2 expression and associated aerobic glycolysis is a general characteristic of all cancer cells, inhibiting PKM2 using the peptides of the invention is expected to provide a global cancer therapy effective against a wide variety of cancer, without damaging normal cells which express PKM1.

It is also believed that Poly (ADP-ribose) polymerase (PARP) may play a role in increasing the potency of the peptides of the present invention. The main role of PARP is to detect and initiate an immediate cellular response to DNA damage, and more specifically, to single-strand DNA breaks (SSB) by signalling the enzymatic machinery involved in SSB repair. Once PARP detects a SSB, it binds to the DNA, undergoes a structural change, and begins the synthesis of a polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) chain, which acts as a signal for the other DNA-repairing enzymes. After repairing, the PAR chains are degraded via Poly(ADP-ribose) glycohydrolase (PARG). Significant DNA damage is commonly seen in cancer cells, and expectedly, upregulation of PARP-1 has been described in many tumour types.

NAD* is required as a substrate for generating ADP-ribose monomers. Normal cells respond to DNA damage by promoting caspase-induced cleavage of PARP, thus inhibiting poly (ADPribosyl ation) and allowing sufficient NADI-to generate the ATP that is necessary for apoptosis, through glycolysis. However, overactivation of PARP, as found in cancer cells with significant DNA damage, may deplete the stores of cellular NAD* and cause further ATP depletion. Therefore, PARP activation in cancer cells may exacerbate the ATP-reducing effects of the peptides of the present invention and facilitate necrosis. Accordingly, a combination of a PA RP agonist and a peptide of the invention may serve as an effective therapeutic agent in the treatment of cancer.

The described and illustrated embodiments are to be considered as illustrative and not restrictive in character with it being understood that only the preferred embodiments have been shown and described, and that all changes and modifications that come within the scope of the inventions as defined in the accompanying claims are desired to be protected.

Examples

Example 1

A cyclic peptide according to the invention (Dac-Arg-Sar-Nal-Arg-Nal-Val-Trp-Trp-ArgArg-Trp-Trp-Arg-Arg) (HILR-56) (SEQ ID NO:9) was synthesised using standard automated methods and cyclised using standard chemical coupling agents. The cytotoxicity of the peptide was tested against human lung cancer (NCI-H460) cells using established methods.

The following protocol was used: 1) NCI-H460 cells were grown in Ham's F12 media supplemented with 10% FBS.

2) Cells were harvested and seeded into 96-well plates at 500 cells/well.

3) The peptide was diluted from DMSO stock solutions from a highest concentration of 500 RM. The final DMSO concentration was constant at 1% (v/v).

4) Diluted peptide samples were incubated in media for 20 minutes in the presence and absence of Nanocin-ProTM (Tecrea) prior to addition to cells. Nanocin-ProTM was used at a final assay concentration of 0.2 Rl/well. Nanocin ProTM is a commercially available nanoparticle reagent which enables improved internalisation of hiomolecules including peptides.

5) Cells were grown in the presence of peptide for 96 hours at 37°C, 5% CO2 in a humidified atmosphere.

6) After 96 hours, Alamar blue 10% (v/v) was added, incubated for a further 4 hours and fluorescent product detected using a BMG FLUOstar plate reader.

7) Media only background readings were subtracted before data was analysed using a 4-parameter logistic equation in GraphPad Prism.

The results are illustrated in Figure 5. HILR-56 exhibited strong cytotoxic activity against the lung cancer cells, with an LD50 of approximately 100nM. This is the lowest LD50 value that has been observed for any PRGPRP peptide variant.

Example 2 -Synergy

The inventor has determined that the active region and cassette region of the cyclic peptides of the present invention act synergistically to provide unexpectedly enhanced cytotoxicity effects. By way of example, approximate LD50 values (i.e. the concentration of peptide which results in 50% cell killing of cells) of cyclic PRGPRP variants with respect to HL460 non-small cell lung cancer cell lines were determined using a comparable protocol to Example 1. The cytotoxicity of the PRGPRP peptide was tested against the RT112 human bladder cancer cell line using a comparable protocol to Example 1 and LD50 values determined. The inventor has established that RT112 and HL460 cell lines respond in the same way to PRGPRP-based peptides. Therefore, comparable results may be expected when using HL640 cells. The results are provided below and illustrated in accompanying Figures 1 to 5.

Table 1 -LD50 values of cyclic PRGPRP and associated variants Cyclic Peptide sequence Peptide SEQ ID LD50 without Nanocin-Pro TM LD50 with NanocinProTM name/Figure reference NO.

Pro-Arg-Arg-Gly-Pro-Arg-Pro N/A; Fig. 1 1 5000p M Estimated to be 70 pM (see explanation below) Pro-Arg-Arg-Gly-Pro-Arg-Pro-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp HILR-25; Fig.2 3 lOpM 0.8pM Cys-Dac-Arg-Sar-Nal-Arg-Nal-Cys HILR-55; Fig.3 11 50pM 5pM Cys-Dac-Arg-Sar-Nap-Arg-Nap-Cys HILR-17; Fig.4 8 200pM 1pM Dac-Arg-Sar-Nal-Arg-N al-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg HILR-56; Fig.5 9 N/A 0.1p M With specific regard to HILR-55 and H1LR-56, Nal was provided in its L-enantiomeric form. Peptides corresponding to HILR-55 in which one or both Nal residues were substituted with the corresponding D-enantiomer were also tested. The LD50 values of the substituted peptides were not significantly different (data not shown).

Table 1 illustrates that the average enhancement in cytotoxicity of the cyclic PRGPRP variants obtained with 0.2% (v/v) Nanocin-ProTM (determined by the reduction in LD50 values) is approximately 73-fold ((10-fold increase for HILR-25+10-fold increase for H1LR-55 +200-fold increase for HILR-17) divided by 3). It may, therefore, reasonably be estimated that the LD50 value of PRGPRP in the presence of Nanocin-ProTM would decrease by 73-fold to approximately 70pM. The above data illustrate that addition of the Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ ID NO:6) cassette to the PRGPRP peptide would be expected to result in approximately a 90-fold increase in cytotoxicity in the presence of Nanocin-ProTM (compare Pro-Arg-Arg-Gly-Pro-Arg-Pro (SEQ ID NO:1) and Pro-Arg-Arg-Gly-Pro-Arg-Pro-Val-TrpTrp-Arg-Arg-Tip-Trp-Arg-Arg-Trp-Tip (SEQ ID NO:3)). Substitution of the proline and glycine residues in the PRGPRP peptide with more hydrophobic amino acid residues according to the present invention would be expected to result in approximately a 14-to 70-fold increase in cytotoxicity in the presence of Nanoci n-Pro cm (compare Pro-A rg-A rg-Gly-Pro-A rg-Pro (SEQ ID NO:1), Cys-Dac-Arg-Sar-Nal-Arg-Nal-Cys (SEQ ID NO:11) and Cys-Dac-Arg-SarNap-Arg-Nap-Cys (SEQ ID NO:8)). Thus, one would reasonably expect up to approximately a 100-to 160-fold increase in cytotoxicity through combined substitution of proline and glycine residues in the PRGPRP peptide with more hydrophobic amino acid residues, and addition of the Val-Trp-Trp-Arg-Arg-Tip-Tip-Arg-Arg cassette. However, what is in fact unexpectedly observed with the peptides of the present invention is a 1000-fold improvement in cytotoxicity in the presence of Nanocin-ProTM (compare Pro-Arg-Arg-Gly-Pro-Arg-Pro (SEQ ID NO:1), and Dac-Arg-Sar-Nal-Arg-Nat-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg (SEQ TD NO: 9)), indicating a synergistic effect.

LIST OF ARTIFICIAL PEPTIDE SEQUENCES

SEQ ID NO:1 Pro-Arg-Gly-Pro-Arg-Pro SEQ ID NO:2 (HILR-001/THR523) Pro-Arg-Gly-Pro-Arg-Pro-Val-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu SEQ ID NO:3 (HILR-25) Pro-Arg-Gly-Pro-Arg-Pro-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp SEQ ID NO:4 Dac-Arg-Sar-Nap-Arg-Nap SEQ ID NO:5 Dac-Arg-Sar-dmPro-Arg-Nap SEQ ID NO:6 (cassette region of HILR-56) Val -Trp-Trp-A rg-A rg-Trp-Trp-A rg-A rg SEQ ID NO:7 Cys-Dac-Arg-Sar-Nal-Arg-Nal-Cys SEQ ID NO:8 (HILR-17) Cys-Dac-Arg-Sar-Nap-Arg-Nap-Cys SEQ ID NO:9 (HILR-56) Dac-Arg-Sar-Nal-Arg-Nal-Val-Trp-Trp-Arg-Arg-Tip-Tip-Arg-Arg SEQ ID NO:10 Dac-Arg-Sar-Nal-Arg-Nal SEQ ID NO:11 Val-Trp-Tip-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Ttp SEQ ID NO:12 Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg

Claims

Claims 1. A cyclic peptide comprising an active region and a cassette region, wherein the active 1 region comprises the amino acid sequence Xx2x3 x4x5,z A6 and a cassette region which comprises the amino acid sequence Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg, or a salt thereof, wherein: X' and X5 arc argininc; and wherein: XI, X3, X4 and X6 are any non-polar amino acid selected from (7-methoxy-coumarin4-yl)-Ala-OH (Dac), sarcosinc (Sar), 3-amino-3-(2-napthyl) propionic acid (Nap), 5,5-dimethylprol ine (dmPro), and 3-(2-Naphthyl)-alaninc (Nal).
2. The peptide according to claim 1, wherein X' is Dac.
3. The peptide according to claim 1 or claim 2, wherein X3 is Sar.
4. The peptide according to any of claims I to 3, wherein X4 and X6 are selected from Nap and Nal.
5. The pcptidc according to claim 4, wherein X4 and X6 arc Nal.
6. The peptide according to claim 5, wherein Nal is 3-(2-Naphthyl)-L-alanine.
7. The pcptidc according to any of claims 1 to 6, wherein the active region of the peptide comprises the amino acid sequence: Dac-Arg-Sar-Nal-Arg-Nal.
8. The peptide according to claim 7, wherein the active region of the peptide consists of the amino acid sequence: Dac-Arg-Sar-Nal-Arg-Nal.
9. The peptide according to any preceding claim, wherein the peptide comprises the sequence: Dac-Arg-Sar-Nal-Arg-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg.
10. The peptide according to claim 8, wherein the peptide consists of the sequence: DacArg-Sar-Nal-Arg-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg.
11. The peptide according to any of claims 1 to 9, wherein the peptide comprises the sequence: Cys-Dac-Arg-Sar-Nal-Ara-Nal-Val-Trp-Trp-Arg-Arg-Trp-Trp-Arg-ArgCys.
12. The peptide according to claim 11, wherein the peptide is cyclised by reacting two thiol groups in the N-and C-terminal cysteine residues with a di-henzyl bromide linker.
13. A formulation comprising the peptide according to any preceding claim and an agent for enhancing the delivery and/or uptake of the peptide into a cell.
14. A formulation according to claim 13. wherein the agent comprises nanoparticles formed of one or more polymers.
15. A pharmaceutical composition comprising the peptide or formulation according to any preceding claim and a pharmaceutical acceptable carrier, diluent or excipient.
16. The pharmaceutical composition of claim 15 wherein the composition comprises a further therapeutic agent.
17. The peptide of any one of claims 1 to 12, the formulation of claim 13 or claim 14, or the pharmaceutical composition of any one of claims 15 or 16 for use in medicine.
18. The peptide of any one of claims 1 to 12. the formulation of claim 13 or claim 14,or the pharmaceutical composition of any one of claims 15 or 16 for use in the treatment of cancer.
19. The peptide, formulation or pharmaceutical composition for use according to claim 18 wherein the use comprises administering the compound or composition with a further therapeutic agent.
20. The peptide, formulation or pharmaceutical composition for use according to claim 18 or claim 19, wherein the peptide, formulation or composition is to be used in a treatment regime further comprising the use of radiation therapy and/or surgery.
21. The peptide, formulation or pharmaceutical composition for use according to any one of claims 18 to 20, wherein the cancer comprises one or more of breast cancer, prostate cancer, colorectal cancer, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer, head and neck cancer, stomach cancer, pancreatic cancer, oesophageal cancer, small cell lung cancer, non-small cell lung cancer, multiple myeloma, melanoma, neuroblastoma, leukaemia, lymphoma, sarcoma or glioma.