C-RAF inhibitors

Abstract

The invention relates to kinase inhibitors, and specifically to inhibitors of the kinase c-RAF, comprising a cyclic peptide component capable of inhibiting complex formation between c-RAF and phosphodiesterase-8A (PDE8A). The inhibitors find use in various medical applications including the treatment of cancer, and in particular cancers which carry gain-of-function mutations in Ras (e.g. KRAS, NRAS or HRAS) and / or RAF mutations (e.g. mutations in A-RAF, B-RAF or c-RAF / RAF1).

Description

[0001] c-RAF Inhibitors

[0002] This application claims priority from GB 2403651.9, filed 13 March 2024, the contents and elements of which are herein incorporated by reference for all purposes.

[0003] Field of the Invention

[0004] The present invention relates to kinase inhibitors and their medical uses, in particular to compounds capable of inhibiting the kinase c-RAF, by inhibiting complex formation between c-RAF and phosphodiesterase-8A (PDE8A).

[0005] Background

[0006] RAS is an oncogene family (K / H / N-RAS) central to regulating normal cell growth and division. Overactivating mutations in RAS occur in > 20% of all human cancers, acting as a master regulator that promotes cancer progression [1-2]. Cancers harbouring an activating RAS mutation are often highly- treatment resistant, presenting patients with poor overall survival rates and increased incidence of metastasis. As mutant RAS drives cancer progression through uncontrolled cell growth, proliferation and migration, many existing and emerging therapies aim to target RAS and / or downstream RAS-effector proteins, such as c-RAF. c-RAF regulates cancer cell proliferation, migration, and survival through multiple kinase-dependent and kinase-independent mechanisms [3-6]. RAS mutant cancers are frequently c-RAF “addicted” and genetic silencing of c-RAF abolishes tumour growth in highly aggressive treatment resistant GEM models of KRAS-driven lung and pancreatic cancer; primarily through kinase- independent mechanisms (i.e., not dependent upon MARK signalling) [7-10]. Not only is c-RAF inhibition well tolerated in these models; related anti-cancer activities are further enhanced by combination with EGFR inhibition, indicative of a synergistic strategy to treating KRAS driven cancer. Current pharmacological approaches aimed at inhibiting c-RAF typically induce intolerable side-effects due to off- target activities, e.g. Sorafenib [11-12]. Moreover, these therapies are only capable of inhibiting c-RAF kinase activity. No c-RAF-specific therapeutics have been developed that are capable of inhibiting c-RAF beyond its kinase-dependent functions. Thus c-RAF represents an established target for cancer drug discovery that remains underexploited

[0013] .

[0007] Phosphodiesterase-8A (PDE8A) has been shown to bind to c-RAF and protect it from inhibitory phosphorylation by protein kinase A (PKA). A peptide derived from the PDE8A sequence is capable of disrupting this interaction, thus increasing the susceptibility of c-RAF to inhibition by PKA and consequently reducing activation of the ERK pathway by c-RAF

[0014] . Cell-permeable versions of this peptide have been described which show anti-proliferative activity [14; 29; WO 2020 / 163771 A1].

[0008] The present invention builds further on that work. Summary of the Invention

[0009] The present invention provides a c-RAF inhibitor, comprising a cyclic component P which is capable of binding to c-RAF and inhibiting interaction between c-RAF and PDE8A, wherein said cyclic peptide component is:

[0010] (i) a cyclic peptide comprising:

[0011] (a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or an all-D or retro-inverso form thereof; or

[0012] (b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or a retro-inverso form thereof;

[0013] (ii) a peptide differing from (i) by no more than 5 substitutions; or

[0014] (iii) a peptoid or N-methyl version of (i) or (ii); or a pharmaceutically acceptable salt thereof.

[0015] The peptides having the sequences of SEQ ID NOs: 1 and 2 correspond to amino acid residues 454-464 of human PDE8A. Thus, the individual positions within cyclic component P can be numbered linearly from 1 to 11 , or alternatively can be given the numbering of the corresponding position from 454 to 464 within the PDE8A sequence as follows:

[0016] For convenience and consistency, linear numbering will be used in this specification where possible.

[0017] If an additional residue is added at the C-terminus, it may be designated as position 465.

[0018] In the context of retro-inverso peptides, the same numbering can still be used. However, in that case, it should be understood that the numbering should be read from C-terminus to N-terminus, instead of the more conventional N-terminus to C-terminus. As previously identified

[0014] , but without wishing to be bound by any particular theory, it is believed that the residues R1 , E7 and Y8 (linear numbering) may be particularly important for interaction with c-RAF. Consequently, it may be desirable that P comprises one, two or all three of Arg (R) at position 1 , Glu (E) at position 7 and Tyr (Y) at position 8 (or their D-form, N-methyl or N-substituted glycine analogues or equivalents). Preferably P includes all three of Arg (R) at position 1 , Glu (E) at position 7 and Tyr (Y) at position 8, or their D-form, N-methyl, or N-substituted glycine analogues. Typically, the side chains of these residues are not involved in cyclisation, or conjugation to other moieties (such as a cell penetrating moiety Z, as described in more detail below).

[0019] Residues R2 and L10 may also engage in significant interactions with c-RAF. Thus it may also be desirable that P comprises Arg (R) at position 2 and / or Leu (L) at position 10, or their D, N-methyl, or N- substituted glycine equivalents. Again, the side chains of these residues are typically not involved in cyclisation, or conjugation to other moieties (such as a cell penetrating moiety Z, as described in more detail below).

[0020] For example, P may include all of R1 , R2, E7, Y8 and L10, or their D, N-methyl, or N-substituted glycine equivalents.

[0021] Typically, the cyclic component P is 11 or 12 residues in length. By “residue” is meant a residue of an amino acid (whether in L or D configuration, and including N-methylated amino acids) or a residue of an N-substituted glycine, as appropriate. Indeed an N-substituted glycine may itself be regarded as an amino acid residue. Typically, while substitutions are permitted as compared to the reference sequences RRLSGNEYVLS (SEQ ID NO: 1) or RRIsGnEYVLS (SEQ ID NO: 2), insertions or deletions are not.

[0022] Thus, where the cyclic component P is 12 residues in length, the additional residue is present at the N- or C-terminus, and so may be designated as position -1 or position 12 relative to the reference sequence.

[0023] Preferably, where the cyclic component P is 12 residues in length, the additional residue is linked via its side chain to a heterologous moiety as described in more detail below.

[0024] Cyclisation may be performed in a number of ways, via the free functional groups at the N-terminus (free amino group) and C-terminus (free carboxylic acid group) of the backbone, via functional groups of side chains, or via a combination of the two.

[0025] The N-terminus or C-terminus of P as described herein refers to the N-terminus or C-terminus, respectively, of a corresponding linear peptide with free terminal groups, such as before cyclisation to form the cyclic component P. For example, an N-terminal amino group of P corresponds to the peptide backbone amino group at residue 1 (linear numbering), and a C-terminal carboxy group of P corresponds to the peptide backbone carboxy group at residue 11 (linear numbering).

[0026] Linkage may be by direct reaction of the relevant functional groups of the peptide. Alternatively, linkage between two or more functional groups may occur via a scaffold moiety. A scaffold moiety has two or more reactive groups, each being capable of forming covalent bonds with respective ones of the peptide functional groups, e.g. terminal or side chain functional groups. A scaffold moiety may be bifunctional, trifunctional, or even higher order, having the appropriate number of reactive groups, each capable of forming covalent bonds with a corresponding peptide functional group, and so may mediate linkage between two, three or even more functional groups. Thus a bifunctional scaffold moiety has two reactive groups, and so is capable of forming a link between two peptide functional groups, e.g. between two side chain functional groups, between two terminal functional groups, or between a side chain functional group and a terminal functional group. A trifunctional scaffold moiety has three reactive groups, and so is capable of forming a link between three peptide functional groups. For example, a trifunctional scaffold moiety may form a link between two terminal functional groups and one side chain functional group. (It will be appreciated that other arrangements are possible, such as a trifunctional scaffold moiety forming a link between two side chain functional groups and one terminal functional group.)

[0027] Thus, cyclisation may involve the formation of covalent bonds between at least:

[0028] - an N-terminal functional group of the backbone (e.g. the N-terminal amino group) and a C-terminal functional group of the backbone (e.g. the C-terminal carboxy group), e.g. to form a peptide bond (so- called “head-to-tail” or “backbone” cyclisation);

[0029] - a side chain functional group (e.g. a carboxylic acid functional group, e.g. of a residue at or near the C- terminus) and an N-terminal functional group of the backbone, such as the N-terminal amino group of the backbone;

[0030] - a side chain functional group (e.g. an amine functional group, e.g. of a residue at or near the N- terminus) and a C-terminal functional group of the backbone, such as the C-terminal carboxyl group of the backbone; or

[0031] - two side chain functional groups, typically a side chain of a residue near the N-terminus and a side chain of a residue at or near the C-terminus.

[0032] - N- and C-terminal functional groups and one side chain functional group, via a trifunctional scaffold moiety;

[0033] - two side chain functional groups and an N- or C-terminal functional group, via a trifunctional scaffold moiety.

[0034] Linkage may be by direct reaction of the relevant peptide functional groups. Alternatively, linkage may occur via a scaffold moiety. A scaffold moiety has two or more reactive groups, each being capable of forming covalent bonds with respective ones of the peptide functional groups, e.g. terminal or side chain functional groups. Thus a scaffold moiety may be bifunctional, trifunctional, or even higher order, having the appropriate number of reactive groups, each capable of forming covalent bonds with a corresponding peptide functional group. Thus a bifunctional scaffold moiety has two reactive groups, and thus is capable of forming a link between two peptide functional groups, e.g. between two side chain functional groups, between two terminal functional groups, or between a side chain functional group and a terminal functional group. A trifunctional scaffold moiety has three reactive groups, and thus is capable of forming a link between three peptide functional groups. A preferred arrangement may be for a trifunctional scaffold moiety to form a link between two terminal functional groups and one side chain functional group, although it will be appreciated that other arrangements are possible, such as a trifunctional scaffold moiety forming a link between two side chain functional groups and one terminal functional group.

[0035] A trifunctional scaffold moiety may comprise a group of Formula (S1) or (S2), where represents a point of attachment to P, such as an N-terminal or C-terminal function group or a side chain of P.

[0036] A cyclic component P comprising a trifunctional scaffold moiety of Formula (S1) or (S2) may be formed from a peptide, such as a linear peptide, comprising three thiol-containing amino acid residues (such as cysteine or homocysteine) and a scaffold comprising a tri-halomethyl substituted phenyl group, such as 1 ,3,5-tris(bromomethyl)-benzene or 1 ,3,5-tris(bromomethyl)-2,4,6-trimethyl-benzene.

[0037] In some embodiments, more than two functional groups of P participate in cyclisation, e.g. three functional groups of P, or four functional groups of P. For example, P may contain combinations of the cyclisation options set out above.

[0038] For example, P may contain (i) head-to-tail cyclisation (between the N-terminal amino group and the C- terminal carboxy group of the backbone), and (ii) cyclisation via two side chain functional groups. Alternatively, three or more functional groups of P may be linked to a scaffold moiety, wherein the three or more functional groups are selected from the N-terminal amino group of the backbone, the C-terminal carboxyl group of the backbone, and one or more side chain functional groups, as already noted above.

[0039] As noted above, the residues at positions 1 , 7 and 8 are believed to be required for optimum interaction with c-RAF and so their side chains typically do not participate in cyclisation. It may be desirable that the side chains of the residues at positions 2 and 10 also do not participate in cyclisation although this may be less important.

[0040] A residue whose side chain participates in cyclisation may be designated by the notation 4) or HL Typically, where a pair of residues is involved in cyclisation, one will be designated 4) and the other as HL

[0041] The residues 4> and H^ may be present at any suitable positions within P. In some embodiments, 4> is present at position 5 and H^ is present at position 9, or 4> is present at position 5 and ^P is present at position 11 .

[0042] However, it will be understood that such residues may participate in cyclisation by being covalently linked to other functional groups, such as the terminal backbone amino or carboxyl groups.

[0043] Where the peptide is cyclised between two side chains (e.g. when P contains a residue 4> and a residue MJ), the side chains may form any appropriate type of covalent bond. Examples include a disulfide bond, a thioether bond, and an amide (lactam) bond. An alternative arrangement would be an “all hydrocarbon” bond formed between two residues each having a side chain with a terminal alkene group, via a ringclosing metathesis (RCM) reaction.

[0044] A disulfide bond may be formed between the side chains of two thiol-containing amino acid residues, such as cysteine or homocysteine. Thus 4> and ^P may be Cys or homoCys, for example 4> and ^P may both be Cys.

[0045] An amide (lactam) bond is typically formed between a side chain having a carboxylic acid functional group (e.g. Asp, Glu or Aad) and a side chain having an amine functional group (e.g. Dap, Dab, Orn, Lys or hLys). Thus one of 4> and ^P may be a residue with a side chain having a carboxylic acid functional group (e.g. Asp, Glu or Aad) and the other may be a residue with a side chain having an amine functional group (e.g. Dap, Dab, Orn, Lys or hLys).

[0046] A thioether bond may be formed in a number of different ways, e.g. between a pair of thiol-containing side chains (such as cysteine or homocysteine) to form a thioether rather than a disulfide, between a side chain having a thiol group (such as cysteine or homocysteine) and a side chain having a hydroxyl group (such as serine or threonine), or between a side chain containing a thiol group (such as cysteine or homocysteine) and a side chain containing a halomethylene group, such as a side chain containing a chloromethylene group (such as chloroalanine, or a side chain containing a chloroacetyl group), a bromomethylene group (such as bromoalanine, or a side chain containing a bromoacetyl group), or an iodomethylene group (such as iodoalanine, or a side chain containing an iodoacetyl group). The same structure (a lanthionine bridge; 3,3-thiodialanine) is formed e.g. between a pair of cysteine side chains, between a cysteine side chain and a serine side chain, or between a cysteine side chain and a betachloro alanine side chain.. Alternatively, a cystathionine bridge may be formed between a homocysteine side chain and a serine side chain or a chloroalanine side chain. Thus one of 4) and may be Cys or homoCys and the other may be Cys, homoCys, Ser, Thr or beta-choro alanine. For example, one of 4) and may be Cys and the other may be Cys, Ser or beta-choro alanine.

[0047] Residues having terminal alkene groups suitable for forming an “all hydrocarbon” bond include alphadisubstituted non-proteinogenic amino acids such as "R5" (i.e. (R)-2-Amino-2-methylhept-6-enoic acid), "S5" (i.e. (S)-2-Amino-2-methylhept-6-enoic acid), "R8" (i.e. (R)-2-Amino-2-methyldec-9-enoic acid) and "S8" (i.e. (S)-2-Amino-2-methyldec-9-enoic acid). Pairs of such residues are often used for stabilisation of alpha-helical structures (by so-called “peptide stapling”) but may also be appropriate for cyclisation in the context of the present invention. See Walensky and Bird, J. Med. Chem. 2014, 57, 6275-6288 (dx.doi.org / 10.1021 / jm4011675), as well as McDougall and Jamieson (Stapled Peptides as Potential Therapeutics. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002 / 9780470015902. a0028403, March 2019) and references cited therein.

[0048] Where the peptide is cyclised between a side chain functional group and the N-terminus of the backbone, the functional group of the side chain may react directly with the free N-terminal amine group, e.g. to form an amide linkage. Thus the side chain may have a carboxylic acid functional group (e.g. Asp, Glu or Aad).

[0049] Where the peptide is cyclised between a side chain functional group and the C-terminus of the backbone, the functional group of the side chain may react directly with the free C-terminal carboxylic acid group, e.g. to form an amide linkage. Thus the side chain may have an amine functional group (e.g. Dap, Dab, Orn, Lys or hLys).

[0050] Alternatively, in either case, the terminal backbone functional group may be derivatised to provide an alternative functional group for reaction with the side chain. For example a moiety containing a halomethylene group may be introduced, e.g. at the N-terminus, and form a thioether bond with a thiol- containing side chain, such as a Cys side chain. Examples of suitable moieties containing a halomethylene group include chloroacetyl, bromoacetyl and iodoacetyl, such as chloroacetyl. The cyclised peptide may thus contain a group represented by (*-C(=O)CH2-**) where * represents the point of attachment of the N-terminus, and ** represents the point of attachment of a thiol-containing side chain.

[0051] The c-RAF inhibitor may further comprise a heterologous moiety. For example, the heterologous moiety may be a membrane transit moiety, a pharmacokinetic-modifying moiety, or a proteolysis targeting moiety.

[0052] The heterologous moiety may be peptidic or non-peptidic, and may be linked to the cyclic component P via a terminal backbone functional group of P or via a side chain of P. The heterologous moiety is typically covalently linked to the cyclic component P. Linkage may be direct or via a linker moiety.

[0053] The c-RAF inhibitor may comprise more than one heterologous moiety, e.g. two or even three such moieties. For example, it may comprise a membrane transit agent in combination with a half-life extending moiety or a proteolysis targeting moiety. However, it may be desirable that a given c-RAF inhibitor comprises a maximum of one heterologous moiety.

[0054] A heterologous moiety may be peptidic or non-peptidic. It may be linked to the cyclic component P via a terminal backbone functional group of P or via a side chain of P. When a heterologous moiety is linked to a side chain of P, the residue of P to which it is linked may be designated may be present at any appropriate position of P although positions 5, 9, 11 and 12 (when present) may be preferred, e.g. position 5. Any residue having an appropriate side chain functional group for attachment may be used at the relevant position of P. The side chains of Ser, Cys, Lys or Gin may be especially appropriate.

[0055] A membrane transit moiety is a moiety which promotes transit of the cyclic component P across the plasma membrane of a target cell.

[0056] As noted above, a membrane transit moiety may be covalently linked to the cyclic component P, i.e. the membrane transit moiety is a component of the c-RAF inhibitor molecule. The membrane transit moiety may also be referred to as a cell penetrating moiety.

[0057] Thus the c-RAF inhibitor may comprise a cyclic component P and a cell penetrating moiety Z. P and Z are covalently linked. For example, Z may be linked to a side chain of P, or to the N- or C-terminus of P, depending on how P is cyclised. Linkage may be direct, or via a linker or spacer group.

[0058] The cell penetrating moiety Z may be a peptide. Such peptides are sometimes referred to as a cell penetrating peptide (CPP) or protein transduction domain (PTD). Such peptides are typically 5 to 30 amino acids in length, and typically carry an overall positive charge or are composed entirely of residues with non-polar and / or hydrophobic side chains. They are sometimes categorised as cationic (often characterised by high arginine content), amphipathic (often having high lysine content) or hydrophobic (composed of hydrophobic and non-polar residues).

[0059] Examples of CPPs include:

[0060] GRKKRRQRRRPPQ or GRKKRRQRRR (Tat) RQIKIWFQNRRMKWKK (Penetratin)

[0061] RKKRRRESRKKRRRES (DPV3)

[0062] GRPRESGKKRKRKRLKP (DPV6)

[0063] RVRVFWHIPRLT (ARF (19-31)

[0064] KLALKLALKALKAALKLA (MAP)

[0065] KETWWETWWTEWSQPKKKRKV (Pep-1)

[0066] GALFLGFLGAAGSTMGAWSQPKKKRKV (MPG)

[0067] VSALK (Bip4)

[0068] CSIPPEVKFNPFVYLI (C105Y)

[0069] GIGAVLKVLTTGLPALISWKRKRQQ (Melittin)

[0070] HGLASTLTRWAHYNALIRAF (gH625)

[0071] PLILLRLLRGQF (Pept 1)

[0072] PLIYLRLLRGQF (Pept 2)

[0073] KLWMRWYSPTTRRYG (IW-14)

[0074] GWTLNSAGYLLGKINLKALAALAKKIL (Transportan)

[0075] MGLGLHLLVLAAALQGAKKKRKV (lg(v))

[0076] KLALKLALKALKAALKLA (Amphiphilic model peptide)

[0077] LLIILRRRIRKQAHAHSK (pVEC)

[0078] RRIPNRRPRR (HRSV)

[0079] RLWMRWYSPRTRAYGC

[0080] GWTLNSAGYLLGKINLKALAALAKKIL poly-arginine (e.g. R4-R18, e.g. Rs)

[0081] GR(4-IO)Q (R-TAT)

[0082] RxWx (i.e. repeating units of Arg and Trp) where x is 3-9, e.g. x is 5.

[0083] Longer peptides may also be suitable, such as the cell-penetrating sequences derived from HOX D12 and HOX C12 described for example in WO 2020 / 163771 :

[0084] ARKKRKPYTKQQIAELENEFLVNEFINRQKRKELSNRLNLSDQQVKIWFQNRRMKKKRVV (HOX D12; also known as CellPorter®);

[0085] SRKKRKPYSKLQLAELEGEFLVNEFITRQRRRELSDRLNLSDQQVKIWFQNRRMKKKRLL (HOX C12); and functional fragments and derivatives thereof.

[0086] See for example, Derakhshankhah and Jafari, Biomedicine & Pharmacotherapy 108 (2018) 1090-1096 and Xie at al., Front. Pharmacol., 20 May 2020, Sec. Experimental Pharmacology and Drug Discovery, https: / / doi.org / 10.3389 / fphar.2020.00697.

[0087] Functional fragments or variants of these peptides which retain cell penetrating activity may also be used.

[0088] For example, variants may contain one or more substitutions compared to the sequences shown. Substitutions with D-equivalents of the original amino acids may be particularly desirable. All-D or retro- inverso forms may also be employed.

[0089] Cyclic versions of any of the above sequences may be particularly useful, and may be indicated by the notation “[cyclo]”. They may have increased stability in vivo (e.g. in plasma) as compared to the linear versions. Examples include cyclo[GRKKRRQRRR] and cyclo[Grkkrrqrrr]. Cyclisation may be achieved by any appropriate means, as described elsewhere in this specification, although head-to-tail cyclisation may be preferred. Head-to-tail cyclisation may be indicated by asterisks “*” at the N- and C-terminal residues, e.g. G*RKKRRQRRR* and G*rkkrrqrrr*.

[0090] Particularly useful examples include the peptides GRKKRRQRRR, Grkkrrqrrr, cyclo[GRKKRRQRRR] and cyclo[Grkkrrqrrr], e.g. GRKKRRQRRR, Grkkrrqrrr, G*RKKRRQRRR* and G*rkkrrqrrr*.

[0091] Amoura et al. (Chem. Commun., 2019, 55, 4566-4569; DOI: 10.1039 / c9cc01265f) describe cyclic oligoArg peptides, optionally including one or more hydrophobic moieties.

[0092] Such peptides include a cyclic component comprising or consisting of a sequence X5-11 where each residue X is arginine or a hydrophobic residue, and the peptide contains at least 4 arginine residues and a maximum of 2 hydrophobic residues. The cyclic component may comprise or consist of a sequence CX5-11 or KX5-11 where each residue X is arginine or a hydrophobic residue, and the cyclic component contains at least 4 arginine residues and a maximum of 2 hydrophobic residues. The N-terminal Cys or Lys residue can be useful to participate in the cyclisation process, and subsequently provides a free thiol or amino group respectively for conjugation to P, which may optionally be via a linker.

[0093] Any hydrophobic residues present may be the same or different. Each may, for example, be Phe (F) or Trp (W), a residue of a 2-amino fatty acid derivative, or a residue of an amino acid with a suitable side chain functional group conjugated to a lipophilic moiety as described in more detail below, e.g. a fatty acyl moiety.

[0094] Residues of 2-amino fatty acid derivatives may be particularly preferred. These include residues of 2- amino butanoic acid, 2-amino hexanoic acid, 2-amino octanoic acid, 2-amino decanoic acid, 2-amino dodecanoic acid, 2-amino tetradecanoic acid, 2-amino hexadecanoic acid, 2-amino octadecanoic acid and 2-amino eicosanoic acid. For example, they may be (R,S)-2-amino butanoic acid, (R,S)-2-amino hexanoic acid, (R,S)-2-amino octanoic acid, (R,S)-2-amino decanoic acid, (R,S)-2-amino dodecanoic acid, (R,S)-2-amino tetradecanoic acid, (R,S)-2-amino hexadecanoic acid, (R,S)-2-amino octadecanoic acid and (R,S)-2-amino eicosanoic acid. (R,S)-2-amino tetradecanoic acid may be particularly preferred. When incorporated into the cyclic peptide, they may be designated by the length of the free carbon chain extending from the alpha-carbon of the peptide backbone. Thus (R,S)-2-amino tetradecanoic acid may be referred to as “C12” (or “(C12)”), and so forth. For conjugation of a fatty acyl moiety to the side chain of an amino acid, any amino acid with a suitable side chain functional group may be used. Examples include Lys, Arg, ornithine, Cys, Ser and Thr, although Lys may be particularly preferred. The fatty acyl moiety may, for example, be an acetyl, butanoyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl or eicosanoyl group.

[0095] As indicated above, cyclisation may be achieved by any appropriate means, as described elsewhere in this specification, although head-to-tail cyclisation may be preferred. Head-to-tail cyclisation may be indicated by asterisks “*” at the N- and C-terminal residues. Cyclisation may be achieved, for example, by native chemical ligation, e.g. via an peptide-mpaL thioester precursor or a peptide having a C-terminal alpha-methylcysteine.

[0096] Examples of such CPPs include:

[0097] C*(Cn)R4*

[0098] C*KfR4* (where f indicates conjugation of the Lys side chain amino group to a fatty acyl group, e.g. a dodecanoyl group)

[0099] C*WR4*

[0100] K*(Cn)R4*

[0101] K*KfR4* (where f indicates conjugation of the Lys side chain amino group to a fatty acyl group, e.g. a dodecanoyl group)

[0102] K*WR4*

[0103] In all cases, n is typically 2, 4, 6, 8, 10, 12, 14, 16, 18, e.g. 10, 12 or 14, e.g. 12.

[0104] C*(Cn)R4* and K*(Cn)R4* may be preferred, e.g. C*(CI2)R4* and K*(CI2)R4*. See Anoura et al. (op cit.) and its Supplementary Information for more details. For ease of comparison, C*(CI2)R4* is designated [CI2-R4] in that paper. The cell penetrating moiety may comprise or consist of any of the above.

[0105] Peptoid equivalents of any of the CPPs described herein may also be employed.

[0106] Alternatively, the cell penetrating moiety Z may be a non-peptide moiety. Non-peptide cell penetrating moieties are typically hydrophobic moieties which are able to be inserted into the plasma membrane of a target cell, e.g. fatty acids (such as stearic (octadecanoic) acid, palmitic (hexadecanoic) acid, myristic (tetradecanoic) acid, lauric (dodecanoic) acid, capric (decanoic) acid, caprylic (octanoic) acid, tannic acid, and lipids such as steroids, e.g. cholesterol).

[0107] The cell penetrating moiety Z may be linked to a side chain of one of the residues of P.

[0108] Where the cell penetrating moiety Z is a peptide (or peptoid), it may be attached to P via a functional group of a side chain of Z, or via a functional group at the N- or C-terminus of the backbone of Z, if available (e.g. via the N-terminal amino group). Thus, for example, the linear peptides GRKKRRQRRR and Grkkrrqrrr may be attached via the backbone amino group of the N-terminal glycine residue.

[0109] Attachment via a backbone functional group will not be possible if Z is cyclised head-to-tail, but will be feasible for linear peptides Z and for peptides Z cyclised via residue side chains. Where attachment is via a side chain functional group of Z, any suitable side chain may be utilised. For example, the cyclic peptides cyclo[GRKKRRQRRR] and cyclo[Grkkrrqrrr] (e.g. G*RKKRRQRRR* and G*rkkrrqrrr*) may be attached via the glutamine (Q or q) side chain. A peptide Z may include a residue (such as a Cys or dCys residue) at the N- or C-terminus for this purpose, so as not to interfere with the cell penetrating activity of the rest of the peptide.

[0110] A terminal functional group of P or Z may be derivatised to provide an alternative functional group for reaction with the other one of P or Z.

[0111] For example, a moiety containing a halomethylene group may be introduced at the N-terminus of one of P or Z, and form a thioether bond with a thiol-containing side chain, such as a Cys side chain, in the other one of P or Z. Examples of suitable moieties include chloroacetyl, bromoacetyl and iodoacetyl, such as chloroacetyl. The c-RAF inhibitor may comprise P or Z having an N-terminus that is derivatised with a moiety containing a methylene group, which is attached to the other one of P or Z via a thioether bond at a thiol-containing side chain. For example, where Z is a linear peptide, the c-RAF inhibitor may comprise a group (*-C(=O)CH2-**) where * represents the point of attachment of the N-terminus of Z, and ** represents a cysteine residue in P. Alternatively, the c-RAF inhibitor may comprise a group (*-C(=O)CH2-**) where * represents the point of attachment of the N-terminus of P, and ** represents a cysteine residue in Z. Linkage of Z to P may be by direct reaction of the relevant functional groups. Alternatively, linkage may occur via linker or spacer moieties, such as bifunctional linker groups.

[0112] Z may be linked to P via a linker. A linker is a divalent moiety in which the two free valencies each form part of a single bond to an adjacent atom. A linker may comprise one or more groups selected from: polyalkyleneglycol, such as polyethylene glycol (PEG) and polypropylene glycol; an optionally substituted alkylene, such as optionally substituted C1-20 alkylene, such as optionally substituted C1-12 alkylene, such as optionally substituted C1-6 alkylene. ether (-O-); thioether (-S-); amino (-N(H)-); amide, which may be represented by (-N(RN)-C(=O)-) or (-C(=O)N(RN)-); ester, which may be represented by (-OC(=O)-) or (-C(=O)O-); carbonyl (-C(=O)-); carbamate, which may be represented by (-OC(=O)N(RNA)-) or (-N(RNA)C(=O)O-), carbonate (-OC(=O)O-); and urea (-N(RNB)C(=O)N(RNC)-).

[0113] RN, RNA, RNBand RNCmay each independently be selected from hydrogen, C1-6 alkyl, C5-2o aryl and C5-20 heteroaryl. Preferably, RN, RNA, RNBand RNCare each independent selected from hydrogen and C1-6 alkyl; such as hydrogen, methyl and ethyl; such as hydrogen.

[0114] Where two or more groups are present in the linker at least one of these groups may be alkylene. Where two or more groups selected from ether, thioether, amino, amide, ester, carbonyl, carbamate, carbonate and urea are present, these groups may be separated by alkylene. Thus, where two or more groups selected from ether, thioether, amino, amide, ester, carbonyl, carbamate, carbonate and urea are present, these groups are typically not directly connected to each other.

[0115] Where an alkylene group is substituted, the substituent may be one or more groups selected from an amido group, such as a formamidyl group (-C(=O)NH2); an ester group, such as (-C(=O)OMe) or (-C(=O)OEt); an acyl group, such as formyl (-C(=O)H) or acetyl (-C{=O)Me); and a carboxy group (-C(=O)OH).

[0116] A linker may comprise an amino group, an alkylene group and a carbonyl group, such as a linker derived from 6-aminohexanoic acid (Ahx).

[0117] Preferably, a linker comprises one or more of a polyethylene glycol, an amide and an optionally substituted alkylene group. An optionally substituted alkylene group, where present, may be substituted with an amido group, such as a formamidyl group. A linker may comprise a methylene group and a carbonyl group, such as wherein the linker comprises a group represented by (-C(=O)-CH2-). In these embodiments, the linker may be attached, such as via the methylene group, to a thiol-containing side chain, such as a cysteine side chain.

[0118] A linker may comprise a scaffold moiety as described herein, such as a bifunctional scaffold moiety. A linker may be covalently attached to P or Z at an N-terminal or C-terminal functional group or at an amino acid residue side chain, such as a glutamine (Q or q) side chain. Preferably, a linker is connected at one end to a side chain in one of P or Z, such as the amino group in the side chain of a glutamine residue (Q or q). The linker may be connected at the other end to an N-terminal or C-terminal functional group, or a side chain, of the other one of P or Z, preferably an N-terminal functional group or a side chain, more preferably the N-terminal amino group or the amino group in the side chain of a glutamine (Q or q) residue.

[0119] Where a linker is attached to an N-terminal functional group of P or Z, this may be to an N-terminal amino group. Where a linker is attached to the C-terminus of one of P or Z, this may be to the C-terminal carbonyl group, such as to provide an amide or ester linkage.

[0120] Where P or Z comprises a free terminus which is not involved in cyclisation or linkage to the other one of P or Z, the free terminus may be attached to, or derivatised to form, an end group. Examples at the C-terminus include an amide end group. Examples at the N-terminus include an acyl group, such as an acetyl group.

[0121] Additionally or alternatively, the c-RAF inhibitor may be linked to a pharmacokinetic-modifying moiety, for example, in order to increase solubility and / or half-life in vivo (e.g. in plasma) and / or bioavailability. Such modification is also known to reduce clearance (e.g. renal clearance) of therapeutic proteins and peptides.

[0122] A pharmacokinetic-modifying moiety may comprise a polymeric or lipophilic moiety. Without wishing to be bound by theory, such a moiety may bind to albumin in the bloodstream, thus shielding the molecule from enzymatic degradation or clearance by the liver, which may enhance the half-life of the c-RAF inhibitor in vivo.

[0123] The polymeric moiety is preferably water soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymeric moieties include polyethylene glycol (PEG), homo- or copolymers of PEG, a monomethyl-substituted polymer of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Francis et al., (1998), Int. J. Hematology 68:1-18; Zalipsky (1995), Bioconjugate Chem. 6:150-165; and Delgado et al. (1992), Crit. Rev. Therap. Drug Carrier Syst. 9:249- 304.

[0124] Other suitable polymeric moieties include poly-amino acids such as poly-lysine, poly-aspartic acid and poly-glutamic acid (see for example Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351 ; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57; Tsukada, et al. (1984), J. Natl. Cancer Inst., vol 73,: 721-729; and Pratesi, et al. (1985), Br. J. Cancer, vol. 52: 841 -848).

[0125] The polymeric moiety may be straight-chain or branched. It may have a molecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000 Da.

[0126] The polymeric moiety may be linked to a terminal group or to the side chain of any suitable residue, in the same way as a cell penetrating moiety.

[0127] A lipophilic moiety may comprise a hydrocarbon chain having from 2 to 24 carbon (C) atoms, such as from 6 to 24 carbon atoms, e.g. 6 to 22 carbon atoms, e.g. 10 to 22 C atoms, e.g. 10 to 20 C atoms. Preferably, it has at least 6 C atoms, and preferably it has 20 C atoms or fewer, e.g. 18 C atoms or fewer. For example, the hydrocarbon chain may contain 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. For example, it may contain 8, 10 or 12 carbon atoms.

[0128] Thus the lipophilic moiety may comprise or consist of a group CH3-(CH2)o-22-(CO)-, e.g. CH3-(CH2)4-22- (CO)-, e.g. CH3-(CH2)4-2O-(CO)-, e.g. CH3-(CH2)6-22-(CO)-, e.g. CH3-(CH2)6-2o-(CO)-.

[0129] For example, it may comprise or consist of a group selected from acetyl, hexanoyl, octanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl and eicosanoyl, preferably octanoyl, decanoyl, dodecanoyl, hexadecanoyl, octadecanoyl or eicosanoyl, more preferably octanoyl, decanoyl, dodecanoyl.

[0130] Alternative such groups are derived from long-chain saturated a, m-dicarboxylic acids of formula HOOC- (CH2)O-22-COOH.

[0131] Thus the lipophilic moiety may comprise or consist of a group HOOC-(CH2)o-22-(CO)-, e.g. HOOC-(CH2)4-22-(CO)-, e.g. HOOC-(CH2)4-2O-(CO)-, e.g. HOOC-(CH2)6-22-(CO)-, e.g. HOOC-(CH2)6-2o-(CO)-.

[0132] For example, the lipophilic moiety may comprise or consist of:

[0133] 5-carboxypentanoyl, i.e. HOOC-(CH2)4-(CO)-;

[0134] 7-carboxyheptanoyl, i.e. HOOC-(CH2)e-(CO)-;

[0135] 9-carboxynonanoyl, i.e. HOOC-(CH2)s-(CO)-;

[0136] 11-carboxyundecanoyl, i.e. HOOC-(CH2)IO-(CO)-;

[0137] 13-carboxytridecanoyl, i.e. HOOC-(CH2)i2-(CO)-;

[0138] 15-carboxypentadecanoyl, i.e. HOOC-(CH2)i4-(CO)-;

[0139] 17-carboxyheptadecanoyl, i.e. HOOC-(CH2)i6-(CO)-;

[0140] 19-carboxynonadecanoyl, i.e. HOOC-(CH2)IS-(CO)-; or

[0141] 21-carboxyheneicosanoyl, i.e. HOOC-(CH2)2o-(CO)-.

[0142] Additionally or alternatively, the c-RAF inhibitor may comprise a proteolysis-targeting moiety, such as a ubiquitin E3 ligase ligand, typically associated to the cyclic component P via a linker, e.g. similar to those described in the context of Z. Binding of the cyclic component P to c-RAF may therefore result in targeting of the c-RAF protein for ubiquitination by E3 ligase and its subsequent degradation by selective proteolysis. This may occur in addition to promoting inhibition of c-RAF by dissociation from PDE8A and consequent phosphorylation. Such a bifunctional molecule may be regarded as a “bifunctional degrader “ (BFD) or “proteolysis-targeting chimera” (PROTAC).

[0143] The E3 ligase ligand may be a ligand for any suitable E3 ligase, including cereblon (CRBN), Von-Hippel Lindau (VHL) protein, members of the Inhibitor of Apoptosis Proteins (IAP) family, MDM2, RNF114, DCAF16, DCAF15, KEAP1 and FEM1 B.

[0144] CRBN ligands are often derivatives of pomalidomide, 4-hydroxythalidomide, alkyl-connected thalidomide derivatives, or lenalidomide.

[0145] VHL ligands include HIF-1-alpha-derived peptides typically 5-7 amino acids in length and containing proline or hydroxyproline, such as the peptide ALAPYIP, and small-molecule ligands such as VH0323, VH101 and VH298.

[0146] For reviews of the PROTAC approach in general, and details regarding specific E3 ligase ligands, see Ishida, et al. (2021) SLAS Discovery. 26(4): 484-502 and Bricelj et al. (2021) Front Chem. 9:707317.

[0147] In some embodiments, the cyclic peptide component P is:

[0148] (i) a cyclic peptide consisting of:

[0149] (a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form thereof; or

[0150] (b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4) or a retro-inverso form thereof;

[0151] (ii) a peptide differing from (i) by no more than 5 substitutions; or

[0152] (iii) a peptoid or N-methyl version of (i) or (ii); wherein the side chain of residue when present, forms a covalent bond with a heterologous moiety.

[0153] In any of the embodiments described above, the cyclic peptide component P may have a sequence consisting of:

[0154] X1 -X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12 wherein

[0155] X1 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof

[0156] X2 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof; X3 is any residue, e.g. Leu, Nle or an aromatic residue;

[0157] X4 is any residue, or a residue 4) or

[0158] X5 is any residue, or a residue 4) or

[0159] X6 is any residue, or a residue 4> or

[0160] X7 is Glu or Asp, or a D-form, N-, methyl, or N-substituted glycine analogue thereof;

[0161] X8 is an aromatic residue, e.g. Tyr, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0162] X9 is any residue, or a residue

[0163] X10 is Leu or Nle, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0164] X11 is any residue, or a residue

[0165] X12 is absent or is a residue wherein:

[0166] P contains a maximum of one residue 4> and one residue ^P; the side chain of any residue 4> forms a covalent bond with the side chain of a residue ^P, with the terminal backbone functional group of residue X11 , or with a scaffold moiety; the side chain of any residue ^P forms a covalent bond with the side chain of a residue 4>, with the terminal backbone functional group of residue X1 , or with a scaffold moiety; and the side chain of any residue forms a covalent bond with a heterologous moiety or with a scaffold moiety; and wherein P is:

[0167] (i) a peptide consisting of:

[0168] (a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form of either; or

[0169] (b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4) or a retro-inverso form of either;

[0170] (ii) a peptide differing from (i) by no more than 5 substitutions; or

[0171] (iii) a peptoid version of (i) or (ii).

[0172] In some embodiments:

[0173] X1 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0174] X2 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0175] X3 is Leu, Nle, or an aromatic residue (e.g. Leu, Nle, Phe, Tyr, Trp or His), or a D-form, N-methyl, or N- substituted glycine analogue thereof;

[0176] X4 is Ser, Ala, Gly, Cys, Pro, Gin, Asn, His or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue 4> or

[0177] X5 is Gly, Ala, Cys, Pro, Gin, Asn, Leu, Nle, His, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue 4> or X6 is Asn, Cys, Met, Gin or His, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue 4) or

[0178] X7 is Glu or Asp, e.g. Glu, or a D-form, N-, methyl, or N-substituted glycine analogue thereof;

[0179] X8 is Tyr, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0180] X9 is Vai, Ala, Gly, Cys, Met, Pro, Gin, Asn, Ser, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue

[0181] X10 is Leu or Nle, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0182] X11 is Ser, Ala, Gly, Met, Pro, Gin or His, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue

[0183] X12 is absent or is a residue wherein:

[0184] P contains a maximum of one residue 4) and one residue ^P; the side chain of any residue 4> forms a covalent bond with the side chain of a residue ^P, with the terminal backbone functional group of residue X11 , or with a scaffold moiety; the side chain of any residue ^P forms a covalent bond with the side chain of a residue 4>, with the terminal backbone functional group of residue X1 , or with a scaffold moiety; and the side chain of any residue forms a covalent bond with a heterologous moiety or with a scaffold moiety; and wherein P is:

[0185] (i) a peptide consisting of:

[0186] (a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form of either; or

[0187] (b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4), or a retro-inverso form of either;

[0188] (ii) a peptide differing from (i) by no more than 5 substitutions; or

[0189] (iii) a peptoid version of (i) or (ii).

[0190] In some embodiments:

[0191] X1 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0192] X2 is Arg, a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0193] X3 is Leu or Nle, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0194] X4 is Ser, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue

[0195] X5 is Gly or an N-methyl analogue thereof, or a residue 4> or

[0196] X6 is Asn, or a D-form, N-methyl, or N-substituted glycine analogue thereof,

[0197] X7 is Glu, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0198] X8 is Tyr, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0199] X9 is Vai, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue ^P or X10 is Leu, or a D-form, N-methyl, or N-substituted glycine analogue thereof;

[0200] X11 is Ser, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue

[0201] X12 is absent or is a residue wherein:

[0202] P contains a maximum of one residue 4) and one residue ^P; the side chain of any residue 4) forms a covalent bond with the side chain of a residue ^P, with the terminal backbone functional group of residue X11 , or with a scaffold moiety; the side chain of any residue ^P forms a covalent bond with the side chain of a residue 4>, with the terminal backbone functional group of residue X1 , or with a scaffold moiety; and the side chain of any residue forms a covalent bond with a heterologous moiety or with a scaffold moiety; and wherein P is:

[0203] (i) a peptide consisting of:

[0204] (a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form of either; or

[0205] (b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4), or a retro-inverso form of either;

[0206] (ii) a peptide differing from (i) by no more than 5 substitutions; or

[0207] (iii) a peptoid version of (i) or (ii).

[0208] In any of the above formulae, it may be preferred that P contains one residue 4> and one residue ^P, and that the side chains of residues 4> and ^P form a covalent bond. Additionally or alternatively, P may be cyclised head-to-tail. Thus, in some embodiments where P is cyclised head-to-tail, P contains no residue 4> or residue ^P.

[0209] P may in addition contain one or more residues each of which forms a covalent bond with a heterologous moiety or with a scaffold moiety. It may be desirable that P contains a maximum of one residue e.g. which forms a covalent bond with a heterologous moiety, e.g. with a membrane transit moiety.

[0210] In any of the embodiments described herein, P may differ from the sequence RRLSGNEYVLS or RRIsGnEYVLS by no more than 5 substitutions, no more than 4 substitutions, no more than 3 substitutions, no more than 2 substitutions or no more than one substitution. For example:

[0211] XI is Arg;

[0212] X2 is Arg;

[0213] X3 is Leu, dLeu or Nle;

[0214] X4 is Ser or dSer;

[0215] X5 is Gly or a residue

[0216] X6 is Asn or dAsn;

[0217] X7 is Glu;

[0218] X8 is Tyr;

[0219] X9 is Vai or a residue ^P;

[0220] X10 is Leu;

[0221] XI I is Ser;

[0222] X12 is absent or is a residue

[0223] In some embodiments:

[0224] XI is Arg;

[0225] X2 is Arg;

[0226] X3 is Leu or dLeu;

[0227] X4 is Ser or dSer;

[0228] X5 is a residue

[0229] X6 is Asn or dAsn;

[0230] X7 is Glu;

[0231] X8 is Tyr;

[0232] X9 is Vai;

[0233] X10 is Leu;

[0234] XI I is Ser;

[0235] X12 is absent; and P is cyclised head-to-tail.

[0236] P may have a sequence selected from:

[0237] RRLSGNEYVLS;

[0238] RRLS NEYVLS;

[0239] RRIsGnEYVLS; and

[0240] RRIs nEYVLS; wherein P is cyclised head-to-tail. Thus, these sequences may be shown as:

[0241] R*RLSGNEYVLS*;

[0242] R*RLS NEYVLS*;

[0243] R*RlsGnEYVLS*; and

[0244] R*Rls nEYVLS*.

[0245] The residue is linked to a heterologous moiety, e.g. a cell penetrating moiety. The residue may be a cysteine residue or a D-cysteine residue.

[0246] The cell penetrating moiety may be any suitable cell penetrating moiety, such as any of those described elsewhere in this specification. For example, it may be a cell penetrating peptide, such as GRKKRRQRRR, Grkkrrqrrr, cyclo[GRKKRRQRRR] or cyclo[Grkkrrqrrr], e.g. GRKKRRQRRR, Grkkrrqrrr, G*RKKRRQRRR* or G*rkkrrqrrr*.

[0247] The c-RAF inhibitor may comprise the cyclic component R*RLSCNEYVLS* or R*RlscnEYVLS* and the CPP Grkkrrqrrr, where the CPP is linked to the thiol of the cysteine or D-cysteine side chain. For example it may be the compound DRx-170 as illustrated in Figure 6 or DRx-173 as illustrated in Figure 19.

[0248] In some embodiments:

[0249] XI is Arg;

[0250] X2 is Arg;

[0251] X3 is Leu or dLeu;

[0252] X4 is Ser or dSer;

[0253] X5 is a residue 4);

[0254] X6 is Asn or dAsn;

[0255] X7 is Glu;

[0256] X8 is Tyr;

[0257] X9 is a residue 4L

[0258] X10 is Leu;

[0259] XI I is Ser;

[0260] X12 is absent or is a residue P may have a sequence selected from RRIs JnEY^- LS and RRIsTmEY^-PLS^.

[0261] The residues 4) and may independently be selected from cysteine and D-cysteine. For example, 4) may be D-cysteine and may be cysteine.

[0262] The residue is linked to a heterologous moiety, e.g. a cell penetrating moiety. The residue may be a cysteine residue or a D-cysteine residue, e.g. a cysteine residue.

[0263] P, e.g. the sequence RRIsdJnEY^PLS, may additionally be cyclised head-to-tail. It is believed that the dual cyclisation assists with transit across the plasma membrane and may reduce the need for a heterologous cell penetrating moiety.

[0264] The cell penetrating moiety may be any suitable cell penetrating moiety, such as any of those described elsewhere in this specification. For example, it may be a cell penetrating peptide, such as GRKKRRQRRR, Grkkrrqrrr, cyclo[GRKKRRQRRR] or cyclo[Grkkrrqrrr], e.g. GRKKRRQRRR, Grkkrrqrrr, G*RKKRRQRRR* or G*rkkrrqrrr*.

[0265] The c-RAF inhibitor may comprise the cyclic component RRIsc*nEYC*LSC and the CPP Grkkrrqrrr, where * indicates disulfide cyclisation between the side chains of the internal D-cysteine and cysteine residues, and where the CPP is linked to the thiol side chain of the C-terminal L-cysteine residue. For example the c-RAF inhibitor may be the compound DRx-174, as illustrated in Figure 19.

[0266] The c-RAF inhibitor may comprise the cyclic component R**Rlsc*nEYC*LS** where ** indicates head-to- tail cyclisation and * indicates disulfide cyclisation between the side chains of the D-cysteine and cysteine residues. For example, the c-RAF inhibitor may be the compound DRx-175 as illustrated in Figure 19.

[0267] The invention further provides a c-RAF inhibitor as described herein for use in a method of medical treatment.

[0268] The invention further provides a c-RAF inhibitor as described herein for use in the treatment of a condition characterised by inappropriate activation of the RAS pathway, or susceptible to treatment by inhibition of c-RAF, e.g. a condition characterised by a mutation in a RAS pathway component. The invention further provides the use of a c-RAF inhibitor as described herein for use in the manufacture of a medicament for treatment of such a condition.

[0269] The invention further provides a method of treating such a condition, comprising administering an effective amount of a c-RAF inhibitor as described herein to an individual in need thereof.

[0270] Such conditions include cancer, in particular a cancer which carries a gain-of-function mutation in Ras (e.g. KRAS, NRAS or HRAS), or a cancer carrying a B-Raf mutation, especially those resistant to treatment with B-Raf inhibitors (such as those which also carry Ras mutations).

[0271] The C-RAF-PDE8 interaction is believed to be important in the development or pathogenesis of inflammatory conditions, by contributing to the pro-inflammatory environment. Such conditions include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and related conditions such as microscopic colitis (including collagenous colitis and lymphocytic colitis), diversion colitis and Behcet’s disease. The c-RAF inhibitor of the invention may therefore find use in treatment of such conditions.

[0272] The c-RAF inhibitor of the invention may also find use in treatment of a RASopathy,

[0273] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0274] Summary of the Figures

[0275] Figure 1 : PEP-FOLD 3.5 peptide structure prediction modelling of PDE8A-derived disruptor peptide. (A) R454 - T465 represents a 0-turn with termini 8.6A apart. (B) Thr465 truncation (peptide R454 - S464) increases formation of secondary structure (P-sheet), forming a P-turn with termini 6.4A apart.

[0276] Figure 2: Peptide array screening analysis of the PDE8A binding region(s) on full-length human c- RAF protein. Peptide array showing c-RAF 20mer peptides (proposed binding region(s)) in which human full-length PDE8A-MBP (not MBP only negative control) protein bound (represented by dark spot).

[0277] Representative heat map (the darker the colour, the higher the binding signal). Strongest (primary) binding region V561 - K590, with additional (accessory) binding areas up / downstream. Accessory binding regions contain PKA-specific inhibitor serine residues 43, 233, 259 and 621 . Figure 3: DRx-peptide target engagement with human (GST-fusion) recombinant c-RAF (kinase domain) protein. (A) Binding curve generated from co-incubation of DRx-100F, DRx-110F, DRx-120F and DRx-150F (15-pt DRC: 0.6 nM - 10 pM) with human recombinant c-RAF-kinase domain protein (GST-fusion) (B) DRx-100F, DRx-110F and DRx-120F binding curves normalised to respective maximum fluorescent signal (i.e., peptide - protein binding saturation of 100%). (C) DRx-100F, DRx-110F, DRx- 120F and DRx-150F (15-pt DRC: 0.6 nM - 10 pM) co-incubated with GST protein control. (D) respective binding affinities (Kd) of DRx-peptides. All data represented as MEAN ± SEM (ns, not significant; *“*, P < 0.0001 , N > 4).

[0278] Figure 4: Peptide array screening analysis of G458 point substitution in R454 - L464 peptide. (A) PEP-FOLD3.5 predicted 3D-structure of R454 - L464, with N-terminal R454, C-terminal S464 and central G458 residues highlighted. (B) Full-length human c-RAF (kinase domain, GST fusion) protein binding to R454 - S464 peptides containing point substitution with every other native L-amino acid. Dark spots represent binding. GST overlay represents negative control peptide. Control peptide and G458 (strongest binding signal) highlighted.

[0279] Figure 5: RTCA xCELLigence - linear DRx-peptide inhibitor assessment in PANC1 pancreatic cancer cells. (A) PANC1 cells subjected to a 48-hour treatment with linear DRx-peptide disruptor panel (500 nM). Cell index normalised to treatment time point 0 (nCI) and represented as a % difference of vehicle control nCI at 48 hour time point. (B) bar chart representing slope of nCI growth curve, normalised as a % difference of vehicle control. (C) Table highlighting relevant differences in each DRx-peptide, with respective % nCI inhibition (taken from 48 Hour time point and as a % difference of vehicle). The PDE8 selective enzyme inhibitor PF-04957325 (Vang AG et al. Front Pharmacol. 2016 Aug 23:7:259; Tsai LCL et al. Horm Metab Res. 2012 Sep;44(10):790-4)) was used as a control.

[0280] Figure 6: Chemical structure of DRx-170.

[0281] Figure 7: RTCA xCELLigence - cyclic DRx-peptide inhibitor assessment in PANC1 pancreatic cancer cells. (A) PANC1 cells subjected to a 48-hour treatment with DRx-120 and DRx-170 disruptor peptides (500 nM). Cell index normalised to treatment time point 0 (nCI) and represented as a % difference of vehicle control nCI at 48 hour time point. (B) bar chart representing slope of nCI growth curve, normalised as a % difference of vehicle control. (C) Table highlights respective % nCI inhibition (taken from 48 Hour time point and as a % difference of vehicle). PF-04957325 is a PDE8 selective enzyme inhibitor control. Figure 8: RTCA xCELLigence - cyclic vs. linear DRx-peptide inhibitor assessment in PANC1 pancreatic cancer cells. PANC1 cells subjected to a 60-hour treatment with DRx-170 (A) and DRx-190 (B) disruptor peptides (0.1 , 0.3 ,1 and 3 pM). Cell index normalised to treatment time point 0 (nCI) and represented as a % difference of vehicle control nCI at 60 hour time point. (C) bar chart representing slope of nCI growth curve, normalised as a % difference of vehicle control. Horizontal dashed line at 50% slope of nCI represents 50% growth inhibition and line at 100% represents vehicle control slope of nCI. (D) Respective LoglC50 of DRx-170 and DRx-190. Data represented as MEAN ± SEM, N > 3. * P < 0.05, ** P < 0.01.

[0282] Figure 9: RTCA xCELLigence - cyclic vs. linear DRx-peptide inhibitor assessment in PANC1 pancreatic cancer cells - 10% FBS (high serum conditions). (A) PANC1 cells subjected to a 60-hour treatment with DRx-170 and DRx-190 disruptor peptides (300 nM). Cell index normalised to treatment time point 0 (nCI) and represented as a % difference of vehicle control nCI at 60 hour time point. (B) bar chart representing slope of nCI growth curve, normalised as a % difference of vehicle control. Slope of curve is determined every 12-hours and normalised to respective vehicle control (100%). Data represented as MEAN ± SEM, N = 3. ns - not significant, * P < 0.05, “ P < 0.01 , ““ P < 0.0001 .

[0283] Figure 10: Proximity Ligation Assay - DRx-peptide panel ability to disrupt the endogenous c-RAF - PDE8A protein complex in PANC1 pancreatic cancer cells. (A) Representative image of PANC1 cells treated with vehicle. Central ‘circular’ stain represents DAPI staining of nuclei. Speckled signal within nucleus and cytoplasm represents PLA signal (indicative of individual c-RAF - PDE8A protein complex events). (B) Graph summarising PLA signal measured following treatment, normalised as a % difference of vehicle (100%). Data represented as MEAN ± SEM, N > 3. * P < 0.05. (C) Representative images of PANC1 cells treated individually with DRx-peptide disruptor panel. Scale bar = 10 pm. All treatments were carried out at 1 pM for 4 hours.

[0284] Figure 11 : Protein expression analysis - DRx-peptide panel ability to upregulate PKA -specific phosphorylation of inhibitory c-RAF serine residues in PANC1 pancreatic cancer cells. (A) In cell western assay - DRx-peptide panel ability to modulate level of inhibitory pS259 c-RAF protein expression in PANC1 cells following 4 hour treatment (3 pM). Phospho-c-RAF normalised to housekeeping protein expression (Actin). Western immunoblotting - DRx-170 ability to upregulate PKA-specific inhibitory phosphorylation of c-RAF (pS259 (B), pS43 (C)) following 4, 24 and 72 hour treatments at 0.3 and 3 pM in PANC1 cells (DRx-150 3 pM negative control peptide). Phospho-c-RAF and total c-RAF were normalised to their respective immunoblots housekeeper protein expression (HSP90), followed by normalised to each other (phospho c-RAF / total c-RAF). pSerine phosphorylation normalised as a % difference of vehicle (100%). (B-C) representative western blot images of total c-RAF and HSP90 are shown twice as the same immunoblots were utilised to normalise phospho-serine c-RAF signal. * P <

[0285] 0.05

[0286] Figure 12: PANC1 3D floating spheroid assay (A) PANC1 spheroids treated 5x QOD over a 10 day period with DRx-150 (10 pM), DRx-170 (10 pM), Afatinib (0.5 pM) or Vehicle control (0.2% DMSO).

[0287] Spheroid area represented as a fold-difference of respective Day 0 area. (B) Representative images of PANC1 spheroids at Day 0 and Day 10. Scale bar = 100 pm. Bar graphs of (C) % spheroid area (Day 10) and (D) spheroid viability (Day 10), represented as % difference of vehicle (MEAN ± SEM, n=5, *** P < 0.001 , *“* P < 0.0001).

[0288] Figure 13: Wound healing (scratch) assay - DRx-170 inhibits PANC1 pancreatic cancer cell migration (A) Representative images of PANC1 monolayer with ‘scratch’ at time-point 0-hours, 24-hours and 48-hours post-treatment with DRx-150 (1 pM), DRx-170 (0.1 , 1 and 10 pM), vehicle (scale bar = 500 pm). (B) % Area of gap, normalised to respective time-point 0-hours (MEAN ± SEM, N > 3). (C) Table highlights % gap closure at 24-hours and 48-hours post-treatment, compared to respective 0-hours time point, (ns, not significant; “ P < 0.01).

[0289] Figure 14: RTCA xCELLigence - analysis of DRx-170 against PANC1 cellular adherence. (A) 8-hour reading of relative cell adherence (i.e., normalised cell index represented as a % difference of vehicle control at 8-hours) of PANC1 cells with or without DRx-150, DRx-170, PF-04957325 or Sorafenib treatment (% different of vehicle, MEAN, n=6). (B) Respective MEAN % adherence (% difference of vehicle) and (C) DRx-170 IC50.

[0290] Figure 15: RTCA xCELLigence - DRx-170 anti-cancer evaluation against a panel of RAS-RAF mutant human cancer cell lines. (A) Relative rate of cell-growth (i.e., slope of growth curve over 60- hour period represented as a % difference vehicle) of RAS-RAF human cancer panel treated with 100 nM DRx-170 for 60 hours (% difference of vehicle, MEAN, n=12). (B) Cancer panel details including: cell line name, cancer type, RAS mutant status, RAF / TP53 mutant status and % growth rate inhibition. MT, Mutant; WT, Wild-Type; PDAC, Pancreatic Ductal Adenocarcinoma; NSCLC, Non-Small Cell Lung Cancer; CRC, Colorectal Cancer; OC, Ovarian Adenocarcinoma; MM, Malignant Melanoma; OS, Osteosarcoma

[0291] Figure 16: Peptide array screening analysis of central L456 - N459 substituted with non-native D- amino acids. Full-length human c-RAF (kinase domain, GST fusion) protein binding to R454 - S464 peptides containing substitution of the central L456 - N459 region with non-native D-amino acids. Dark spots represent binding. GST overlay represents negative control peptide. Control peptide highlighted by arrow.

[0292] Figure 17: Peptide array screening point substitution analysis of RRLSCNEYVLS with L-amino acids. Full-length human c-RAF (kinase domain, GST fusion) protein binding to RRLSCNEYVLS-based peptides following point substitution with all L-amino acids. Dark spots represent binding. (A) GST overlay represents negative control peptide. Dark spots indicate non-specific binding of GST. (B) c-RAF overlay (C) Representative heat map presents c-RAF binding to peptides as a % difference of control peptide (100% binding = RRLSCNEYVLS). GST (non-specific) binding subtracted from c-RAF-GST binding signal appropriately.

[0293] Figure 18: In vitro plasma stability assessment of DRx-170 in mice, rat and human. Peptides incubated for up to 2 hours and half-life (T 14; mins) calculated.

[0294] Figure 19: In vitro whole blood stability assessment of DRx-170, DRx-173, DRx-174 and DRx-175 in mice. (A) Peptides incubated for up to 2 hours and half-life (T 14, mins) calculated. ChemDraw structures of (B) DRx-173, (C) DRx-174, (D) DRx-175

[0295] Figure 20: RTCA xCELLigence - analysis of DRx-170 - DRx-175 against PANC1 relative cell growth. PANC1 cells seeded and allowed to grow for 18-24 hours before being treated for a further 48 hours with a 6 point dose-response (0.001 pM - 10 pM) of (A) DRx-170, (B) DRx-173, (C) DRx-174, (D) DRx-175. DRx-170-negative control peptide (3 pM) and vehicle only (< 0.5% DMSO) were used as negative controls. PANC1 cells were incubated and treated in high (10% FBS) serum conditions. Data presented as MEAN ± SEM, where relative rate of cell growth is represented as a % diff of vehicle (N=4- 7).

[0296] Figure 21 : Proximity Ligation Assay - disruption of the endogenous c-RAF - PDE8A protein complex in PANC1 PDAC cells. Representative images of PANC1 cells treated with vehicle (0.25% DMSO), or 10 pM DRx-170-C negative control peptide, DRx-170, DRx-173, DRx-174, DRx-175, MRTX1133 KRAS G12D inhibitor (Biswas, S et al., Immunity 2023 Nov 14;56(11):2570-2583), PF04957325 PDE8 catalytic site inhibitor for 4 Hrs. Solid circular grey shape represents individual cell nuclei. Speckled signal within nucleus and cytoplasm represents PLA signal (indicative of individual c-RAF - PDE8A protein complex formation). n>30 cells per group. Figure 22: Wound healing (scratch) assay - DRx-174 inhibits PANC1 pancreatic cancer cell migration. (A) Representative images of PANC1 monolayer with ‘scratch’ at time-point 0-hours, 24-hours and 48-hours post-treatment with DRx-170-C (1 pM), DRx-174 (1 pM), vehicle (scale bar = 500 pm). (B) % Area of gap, normalised to respective time-point 0-hours (MEAN ± SEM, N > 3). (C) Table highlights % gap closure at 24-hours and 48-hours post-treatment, compared to respective 0-hours time point, (ns, not significant; ** P < 0.01).

[0297] Figure 23: RTCA xCELLigence - analysis of DRx-174 against a panel of KRAS mutant human pancreatic and colorectal cancer cell lines. Each cell line was treated with a 6 point dose response of DRx-174 (0.001 pM to 10 pM) for 48 Hrs (10% FBS, complete medium). Representative log[pM] growth IC50 presented in graph for each cell line. Lineage C (colorectal cancer) and P (pancreatic cancer) highlighted below, alongside KRAS mutational status and relative growth IC50 (pM). HTZ (heterozygous KRAS mutant), HMZ (homozygous KRAS mutant). N>3, MEAN ± SEM.

[0298] Figure 24: RTCA xCELLigence - analysis of DRx-174 and Afatinib combination treatment against (KRAS G12D heterozygous) PANC1 relative cell growth. PANC1 cells treated with a 7 point dose response of Afatinib (± 0.5 pM or 1 pM DRx-174) for 48 Hrs (10% FBS, complete medium). (A) doseresponse curve of monotherapeutic and combination therapy treatments, where relative rate of growth is quantified as a % diff. of vehicle (0.25% DMSO). (B) relative growth IC50s following monotherapeutic vs. combination treatment (LoglC50, pM). N=3, MEAN ± SEM, *, P < 0.05.

[0299] Figure 25: RTCA xCELLigence - analysis of DRx-174 and MRTX1133 (KRAS G12D inhibitor) against (KRAS G12D heterozygous) PANC1 relative pancreatic cancer cell growth. PANC1 cells were treated with vehicle (0.25% DMSO), DRx-174 (0.5 pM), MRTX1133 (1 pM) or combination DRx-174 (0.5 pM) - MRTX1133 (1 pM) for 96 Hrs (10% FBS, complete medium). Normalised cell index (i.e. relative cell growth) curves (left) represented as a % difference of vehicle at 96 Hrs (i.e., 100%).

[0300] Respective slopes (i.e., relative rate of growth) to right of cell index data. N=3, MEAN ± SEM. * P < 0.05,

[0301] ** P < 0.01.

[0302] Figure 26: RTCA xCELLigence - analysis of DRx-174 and MRTX1133 (KRAS G12D inhibitor) against (KRAS G12D homozygous) Panc08.13 relative pancreatic cancer cell growth. Panc08.13 cells were treated with vehicle (0.25% DMSO), DRx-174 (0.1 pM), MRTX1133 (1 pM) or combination DRx-174 (0.1 pM) - MRTX1133 (1 pM) for 96 Hrs (10% FBS, complete medium). Normalised cell index (i.e. relative cell growth) curves (left) represented as a % difference of vehicle at 96 Hrs (i.e., 100%).

[0303] Respective slopes (i.e., relative rate of growth) to right of cell index data. N=3, MEAN ± SEM. * P < 0.05, **** P < 0.0001. Figure 27: RTCA xCELLigence - analysis of DRx-174 and AMG510 (Sotorasib, KRAS G12C inhibitor) against (KRAS G12C homozygous) SW837 relative colorectal cancer cell growth. SW837 cells were treated with vehicle (0.25% DMSO), DRx-174 (0.25 pM), AMG510 (0.01 pM) or combination DRx-174 (0.25 pM) - AMG510 (0.01 pM) for 168 Hrs (10% FBS, complete medium). Normalised cell index (i.e. relative cell growth) curves (left) represented as a % difference of vehicle at 168 Hrs (i.e., 100%). Respective slopes (i.e., relative rate of growth) to right of cell index data. N=3, MEAN ± SEM. **** P < 0.0001.

[0304] Figure 28: RTCA xCELLigence - analysis of DRx-174 and RMC-6236 (Daraxonrasib, multi KRAS mutant inhibitor) against (KRAS G13D homozygous) HCT116 relative colorectal cancer cell growth. HCT116 cells were treated with vehicle (0.25% DMSO), DRx-174 (0.1 pM), RMC-6236 (0.03 pM) or combination DRx-174 (0.1 pM) - RMC-6236 (0.03 pM) for 96 Hrs (10% FBS, complete medium). Normalised cell index (i.e. relative cell growth) curves (left) represented as a % difference of vehicle at 96 Hrs (i.e., 100%). Respective slopes (i.e., relative rate of growth) to right of cell index data. N=3, MEAN ± SEM. * P < 0.05, **** P < 0.0001.

[0305] Figure 29: DRx-174 pharmacokinetics. DRx-174 single intravenous dose (bolus) at 5 mg / kg in a CD-1 mouse and subsequent plasma / muscle DRx-174 concentration quantified. Respective circulating plasma half-life highlighted in table (right). n=3 mice per time-point.

[0306] Figure 30: DRx-174 monotherapy inhibits primary tumour growth in a sub-cutaneous PANC1 (KRAS G12D heterozygous pancreatic cancer) cell derived xenograft (CDX) immunodeficient (NSG, female) mouse model. Treatment(s) began once sub-cutaneous growing (flank) PANC1 tumours reached 50-150 mm3. Mice were treated with DRx-174 (intraperitoneally (IP)) for 28-days, following 14 days without treatment. Full 42-day study curves highlight relative PANC1 tumour volume as a % difference of Day 0 (i.e., normalised tumour volume (nTV) = 100%). Tumour growth n=9 mice per treatment arm, MEAN ± SEM. ##, P < 0.0001 vs. vehicle.

[0307] Figure 31. DRx-174 on-target tumour activity in the sub-cutaneous PANC1 (KRAS G12D heterozygous pancreatic cancer) CDX immunodeficient (NSG, female) mouse model. Western immunoblotting analysis of relative PDE8A1 / 2, c-RAF, pS259 c-RAF, STAT3, pY705 STAT3, GAPDH protein levels in homogenised PANC1 tumours at Day 28 (N=3 tumours) (A) and Day 42 (N=6 tumours) (B) of study (see Figure 30). (C) Table highlights relative % difference (% A protein vs. vehicle) in protein levels, including whether they were statistically significant. Ns, not significant; *, P < 0.05; **, P < 0.01 . Detailed Description of the Invention

[0308] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0309] Throughout the present description and claims the conventional three-letter and one-letter codes for naturally occurring amino acids are used, i.e. (lie), V (Vai), F (Phe), W (Trp), S (Ser), T (Thr), Y (Tyr), N (Asn), Q (Gin), D (Arg), H (His), M (Met), C (Cys) and P (Pro).

[0310] By “naturally occurring” in this context is meant the 20 amino acids encoded by the standard genetic code, sometimes referred to as proteinogenic amino acids.

[0311] Less common or non-naturally occurring amino acids (i.e. amino acids other than the 20 encoded by the standard mammalian genetic code) may be referred to by their full name (e.g. sarcosine, ornithine, etc.) or by frequently employed three- or four-character codes. Examples include: Norleucine (Nle; 2-aminohexanoic acid);

[0312] Orn (ornithine, i.e. 2,5-diaminopentanoic acid);

[0313] Dab (2,4-diaminobutanoic acid; e.g. (2S)-2,4-diaminobutanoic acid);

[0314] Dap (2,3-diaminopropanoic acid; e.g. (2S)-2,3-diaminopropanoic acid);

[0315] Aad (2-aminoadipic acid, e.g. (2S)-2-aminoadipic acid; also known as (2S)-2-aminohexanedioic acid or homo-glutamic acid); hLys (2-amino-7-amino-heptanoic acid, also known as homo-lysine, e.g. (2S)-2-amino-7-amino-heptanoic acid) hCys (homo-cysteine) chloroalanine (beta-chloroalanine; 3-chloroalanine)

[0316] R5 ((R)-2-Amino-2-methylhept-6-enoic acid)

[0317] S5 ( (S)-2-Amino-2-methylhept-6-enoic acid)

[0318] R8 ((R)-2-Amino-2-methyldec-9-enoic acid)

[0319] S8 ((S)-2-Amino-2-methyldec-9-enoic acid)

[0320] N-methylated amino acids may also be employed. Sarcosine (Sar; N-methylg lycine) is one example of an N-methylated amino acid, but N-methylated equivalents of any amino acid (other than proline) may be used. They may be designated [NMe-X] where X is the single letter code, three letter code, or full name of the relevant residue. When using the single letter code, lower case letters are used for amino acid residues of the D- configuration, and upper case letters for amino acid residues of the L-configuration. For non-chiral amino acid residues (e.g. glycine), either may be used. When the three letter code is used, the configuration D- or L- is typically indicated explicitly. If no configuration is shown, the L-configuration should be assumed. For the avoidance of doubt. SEQ ID NOs: 3 and 4 (RRIsGnEYVLS and RRIsGnEYVLS^) contain D- isomers of L456, S457 and N459, rather than an L-isoleucine residue at position 3.

[0321] When applied to a given amino acid sequence, the term "retro-inverso" is used to indicate an alternative form containing the same residues, in the opposite configuration (L or D), and in which the order of the residues from N- to C-terminus is reversed. The term is often used to refer to a reversed-sequence all-D version of a conventional peptide consisting of L-form residues. In the context of the present invention, a “retro-inverso” form of a peptide containing both L- and D-amino acids would have the reverse N- to C- orientation, with L-amino acids exchanged for D-amino acids and D-amino acids exchanged for L-amino- acids. Thus, for example, a retro-inverso version of the sequence RRIsGnEYVLS (SEQ ID NO: 2) would have the sequence sIvyeNGSLrr.

[0322] The N-substituted glycines are closely related to proteinogenic or non-proteinogenic amino acids, but differ in that their side chains are linked to the backbone nitrogen atom rather than to the alpha-carbons (as in conventional amino acids). N-substituted glycine monomers may be designated by the prefix “N-” applied to the designation of the amino acid with the corresponding side chain. Thus, by way of illustration, N-Arg refers to N-substituted glycine with an arginine side chain ((3-guanidinopropylamino)- acetic acid), N-GIn refers to N-substituted glycine with a glutamine sidechain ((2-carbamoyl-ethylamino)- acetic acid), and N-Tyr refers to N-substituted glycine with a tyrosine sidechain (N-(4- hydroxyphenyl)glycine). The meaning of other abbreviations will be apparent from the context.

[0323] The N-substituted glycines are not chiral and so do not typically have L and D enantiomers.

[0324] Illustrative formulae for N-Tyr, N-Arg and N-GIn residues (as part of a longer peptide or peptoid chain) are provided below.

[0325] N-Arg:

[0326] N-GIn:

[0327] The term “peptoid” is used in this specification to designate a peptidomimetic sequence or molecule composed entirely of N-substituted glycine monomers. However, it will be understood that N-substituted glycine units may also be used in combination with conventional amino acids. Molecules containing both conventional amino acids and N-substituted glycine units are considered simply as peptides for the purposes of this specification. It will be understood that the moieties P and X may independently be peptide or peptoid moieties, and that the c-RAF inhibitor may therefore comprise a peptide linked to a peptoid.

[0328] Without wishing to be bound by theory, it is believed that identity of the amino acid side chains at each position may be more important to the peptide’s function than whether they are part of an L amino acid residue, a D amino acid residue, an N-methylated amino acid residue (of L or D configuration), or an N- substituted glycine residue. Thus, for any given side chain, these different forms may be regarded as equivalents of one another, rather than as one being a substitution for another.

[0329] It will be apparent that the terminal functional groups (amino and carboxyl) of the peptide chain of P may participate in cyclisation. Alternatively, either or both may be linked to a heterologous moiety, such as a half-life extending moiety or a cell transit moiety. Alternatively, either or both may be derivatised to increase stability. Thus the N-terminal amino group may be represented by -NH-Y1 , where Y1 may be hydrogen (H), acetyl (Ac) or a heterologous moiety such as a half-life extending moiety or a cell transit moiety. The C-terminal carboxyl group may be represented by -C(O)-Y2, where Y2 may be OH (forming a terminal carboxylic acid group), NH2 (forming a terminal amide), or a heterologous moiety such as a half-life extending moiety or a cell transit moiety. In some embodiments, the cyclic component P comprises only L and / or D amino acids. c-RAF inhibitor

[0330] The 12-mer peptide RRLSGNEYVLST, derived from the human phosphodiesterase-8A (PDE8A) sequence, has been shown to act as an inhibitor of c-RAF (also known as Raf-1) by disrupting the interaction between c-RAF and PDE8A, thus exposing c-RAF to inhibitory phosphorylation by protein kinase A (PKA). Cell-permeable derivatives of that peptide, linked to a stearic acid moiety or the Cell Porter® membrane-transit moiety, have anti-proliferative activity [14; 29; WO 2020 / 163771].

[0331] The inventors have now modelled the interaction of this peptide with the c-RAF molecule and shown that it adopts a hairpin-like conformation when bound to c-RAF. The resulting structure suggested that removal of the C-terminal threonine residue (T465) would lead to an increase in beta-strand secondary structural elements (particularly surrounding R454-L456 and E460-Y461), potentially conferring increased structural rigidity, and would also bring the termini of the peptide closer together, making it more suitable for cyclisation. Cyclisation would be desirable for increasing the stability of the molecule in vivo, since linear peptide therapeutics are notoriously susceptible to degradation.

[0332] Thus the c-RAF inhibitor of the invention is capable of binding to c-RAF and inhibiting interaction between c-RAF and PDE8A. By “inhibiting interaction” is meant inhibiting complex formation between c-RAF and PDE8A molecules, and / or disrupting complexes already formed between c-RAF and PDE8A. c-RAF is a potential therapeutic target in a number of conditions. Principally, it is a target in cancers which carry gain-of-function mutations in Ras (e.g. KRAS, NRAS or HRAS), which account for over 25% of all human cancers. Thus the c-RAF inhibitors described in this specification may find use in the treatment of such cancers. These include: cancers of the central nervous system (CNS), such as glioblastoma multiforme (GBM) and lower grade glioma (LGG); cancers of the head and neck, such as head and neck squamous cell carcinoma (HNSC); endocrine cancers, such as papillary thyroid carcinoma (THCA); anaplastic thyroid carcinoma (THCAA); follicular thyroid carcinoma (THCAF); thoracic cancers, such as lung adenocarcinoma (LUAD); lung squamous cell carcinoma (LUSC); breast cancers, such as breast invasive carcinoma (BRCA); core gastrointestinal (Gl) cancers, such as esophageal carcinoma (ESCA); stomach adenocarcinoma (STAD); small intestine adenocarcinoma (SIAD); colon adenocarcinoma (COAD); rectal adenocarcinoma (READ); accessory gastrointestinal cancers, such as liver hepatocellular carcinoma (LIHC); cholangiocarcinoma (CHOL); gallbladder carcinoma (GBC); pancreatic adenocarcinoma (PAAD); genito-urinary (GU) cancers, such as kidney renal clear cell carcinoma (KIRC); bladder urothelial carcinoma (BLCA); prostate adenocarcinoma (PRAD); gynaecological cancers, such as ovarian serous cystadenocarcinoma; uterine corpus endometrial carcinoma (UCEC); cervical squamous carcinoma and endocervical adenocarcinoma (CESC); skin cancers such as skin cutaneous melanoma (SKCM); haematological cancers, such as acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic myeloid leukemia (CML); plasma cell myeloma (PCM).

[0333] The prevalence of Ras mutations in such cancers is discussed, for example, in Prior et al., Cancer Res 2020;80:2969-74; doi: 10.1158 / 0008-5472. CAN-19-3682.

[0334] Pancreatic cancers (e.g. pancreatic adenocarcinoma) and lung cancers (e.g. lung adenocarcinoma) may be of particular interest.

[0335] Paediatric cancers may also carry such gain-of-function mutations in Ras, and so are also suitable for treatment with the agents described here. These include NRAS-driven neuroblastoma, for example. c-RAF inhibitors may also find use in the treatment of cancers containing B-Raf mutations, especially those resistant to treatment with B-Raf inhibitors (such as those which also carry Ras mutations).

[0336] Cancers carrying so-called type II and type III B-Raf mutations may be particularly suitable. Type I B-Raf mutations are mutations at V600, such as V600E. Type II and III B-Raf mutations may therefore be referred to as non-V600 mutations. Type III mutations are typically accompanied by a Ras mutation. These include melanomas, especially B-Raf inhibitor-resistant melanomas. It is believed that a so-called “paradoxical activation” of ERK signalling may occur in such cancers, as a result of heterodimer formation between B-Raf and c-RAF, and hence c-RAF inhibitors may have therapeutic value.

[0337] The inhibitors of the invention may be used in combination with another anti-cancer therapy. Indeed, they are believed to exhibit synergy with other therapies such as inhibitors of EGFR-family members, especially inhibitors of EGFR (epidermal growth factor receptor) itself inhibitors and RAS inhibitors (e.g., AMG510, MRTX1133, RMC-6236). Inhibitors of EGFR include small molecule inhibitors such as afatinib, gefitinib, erlotinib, brigatinib, lapatinib, icotinib and osimertinib, and monoclonal antibody inhibitors such as cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab. RAS inhibitors include mutant-selective inhibitors such as AMG510 (Sotorasib), MRTX1133, Adagrasib, Garsorasib, Divarasib, JNJ-74699157 and RMC-9805, as well as broader spectrum multi-mutant inhibitors such as RMC-6236, RMC-7977, BI-3706674, LY4066434 and PF-079434040. A review of RAS inhibitors is provided by Isermann et al. (Trends Cancer, 11 (2), 91-116, 2024; doi. org / 10.1016 / j.trecan.2024.11 .009).

[0338] The C-RAF-PDE8 interaction is believed to be important in the development or pathogenesis of inflammatory conditions, by contributing to the pro-inflammatory environment. Such conditions include autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease (including Crohn’s disease and ulcerative colitis) and related conditions such as microscopic colitis (including collagenous colitis and lymphocytic colitis), diversion colitis and Behcet’s disease. Inflammatory bowel disease and its related conditions may be a precursor to development of gastrointestinal cancers, so their treatment may represent prophylaxis of gastrointestinal cancer. The inhibitors described herein may therefore find use in treatment of such conditions.

[0339] Germline mutations in RAS pathway components are implicated in a number of disorders collectively known as RASopathies, which include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NS-ML, previously referred to as LEOPARD syndrome), Noonan syndrome with loose anagen hair (NS-LH), cardiofaciocutaneous syndrome (CFC), Costello syndrome (CS), Legius syndrome (LS), central conducting lymphatic anomalies syndrome (CCLA), SYNGAP1 syndrome, and capillary malformation arteriovenous malformation syndrome (CM-AVM). See Hebron et al., Disease Models & Mechanisms (2022) 15, dmm049107. doi:10.1242 / dmm.049107. The inhibitors of the invention may therefore also find use in the treatment of a RASopathy.

[0340] Pharmaceutical Compositions

[0341] The c-RAF inhibitors described in this specification can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes or topical application.

[0342] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0343] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and / or other additives may be included, as required.

[0344] Administration is preferably in a “prophylactically effective amount” or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example Remington’s Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

[0345] When the c-RAF inhibitor does not comprise a membrane transit moiety, it may be provided in non- covalent association with a membrane transit agent, e.g. in a mixture with a membrane transit agent, or encapsulated within a membrane transit agent. For example, the agent may be a liposome-forming agent, and the c-RAF inhibitor may be encapsulated within a liposome or liposomes.

[0346] Salts and Solvates

[0347] The agents described herein may be provided in the form of a suitable salt, such as pharmaceutically acceptable salts. Suitable salts include those formed with organic or inorganic acids or bases. Pharmaceutically acceptable acid addition salts include those formed with hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycollic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p- toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, and isethionic acids. Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutical acceptable salts. Pharmaceutically acceptable salts with bases include ammonium salts, alkali metal salts, for example potassium and sodium salts, alkaline earth metal salts, for example calcium and magnesium salts, and salts with organic bases, for example dicyclohexylamine and N-methyl-D-glucomine.

[0348] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. Such complexes are known as "solvates". For example, a complex with water is known as a "hydrate". The present invention provides solvates of compounds of the invention.

[0349] Peptide Synthesis

[0350] The c-RAF inhibitors of the invention may be made by any suitable technique for making peptides, including but not limited to conventional methodology, for example, synthesis from individual amino acids, especially step-wise synthesis using an automatic peptide synthesizer; modification of native peptides; or recombinant manufacturing techniques.

[0351] In general, it will be preferred to synthesise the inhibitors by means of solid-phase or liquid-phase peptide synthesis methodology. In this context, reference may be made, for example, to Fields, G.B. et al., 2002, “Principles and practice of solid-phase peptide synthesis”. In: Synthetic Peptides (2nd Edition). The skilled person will be well aware of suitable methodologies.

[0352] The individual peptide components (P, and Z where appropriate) may alternatively be produced by recombinant methods, e.g. by expressing a precursor peptide sequence from a nucleic acid construct that encodes the precursor peptide, recovering the expression product, and modifying the precursor peptide to yield a c-RAF inhibitor of the invention. Expression may be performed in a host cell or in a cell-free expression system. When producing the precursor peptide from cells, it may be convenient that the expression product is secreted into the culture medium.

[0353] Examples

[0354] Materials and Methods

[0355] Antibodies and Chemicals:

[0356] Primary antibodies included ERK1 / 2 (Cell Signaling, 4696), pERK1 / 2 (Cell Signaling, 9101), PDE8A (Protein-Tech, 13956-1 -AP), c-RAF (Cell Signaling, 9422), c-RAF (Sigma, R2404) c-RAF pS338 (Abeam, 150365), c-RAF pS43 (Abeam, ab150365), c-RAF pS259 (Cell Signaling, 9421), STAT3 (Cell Signalling, 9139), pY705 STAT3 (Cell Signalling, 9145), HSP90 (Santa Cruz, sc-7947). IRDye (LI-COR) secondary antibodies included 800CW Donkey anti-Rabbit IgG (926-32213) and 680RD Donkey anti-mouse IgG (926-68072). Alexa Fluor secondary antibodies included Donkey anti-mouse 647nm (ThermoFisher Scientific, A-31571) and Donkey anti-rabbit 488nm (ThermoFisher Scientific, A-48269). Antibodies were diluted in Intercept T20 TBS antibody diluent (LI-COR). Stock concentrations of MRTX1133 (MCE, HY- 134813), AMG510 (MCE, HY-114277), RMC-6236 (MCE, HY-148428), Afatinib dimaleate (R&D Systems,

[0357] 6812), PPL-008C, DP-1 and all DRx-peptides were diluted in 100% DMSO to 10 mM.

[0358] Table 1 : PDE8A-Derived Peptides

[0359] Peptide Array:

[0360] Peptide array experiments were performed by automatic SPOT synthesis as described [14, 27]. Human c-RAF peptides were synthesised onto continuous cellulose membrane supports via 9- fluorenylmethyloxycarbonyl chemistry (Fmoc) using the MultiPep 2 Robot (OEM). A far western blot approach was utilised to detect PDE8A-MBP binding, whereby c-RAF arrays were (i) blocked for 1 hour at room temperature, (ii) incubated overnight at 4°C in 0.1 pM PDE8A1-MBP (diluted in 1X TBS, 5% glycerol, pH 7.4) (Brown, et al. 2013), (iii) incubated for 1 hour at room temperature in aMBP-HRP primary antibody (1 :1000, Abeam: ab49923) and (iv) visualised via ECL detection utilising the C -Digit Blot Scanner (LI-COR). MBP was used as a negative protein control. The same approach was taken to detect c-RAF-GST binding to PDE8A arrays, incubating with 0.1 pM c-RAF-GST protein or GST protein only, followed by incubation with a aGST-HRP primary antibody (1 :5000, Sigma, A7340) and ECL detection. Table 2: Fluorescently labelled (C-terminal 5-FITC) PDE8-Derived Peptides

[0361] Peptide Structure

[0362] Fluorescent Ligand-Based Binding Assay:

[0363] A fluorescent based target engagement assay was utilised to measure the binding affinity of PDE8- derived peptides (C-terminal FITC labelled, see Table 2) with human recombinant c-RAF kinase domain protein (14-352, Merck). Glutathione coated wells of a pre-blocked, black, clear bottom, 96-well plate (15340 - ThermoFisher Scientific) were incubated with c-RAF kinase domain protein or GST protein (10 ng / well) and incubated overnight at 4°C. Wells were then incubated with increasing concentrations (15- point dose response) of FITC-labelled peptide (0.6 nM - 10 pM) for 2 Hours at room temperature. Excess protein / peptide were removed following each incubation step by washing 3 times in 1X TBS-T. Protein and peptides were diluted in protein buffer (200 mM NaCI, 50 mM Tris, 5 mM DTT, 5% Glycerol, protease cocktail inhibitor tablet (Roche), pH 7.5). FITC-peptide binding to c-RAF protein was measured using a Tristar 5 multimode microplate reader (Berthold Technologies). Binding affinities were measured via nonlinear regression analysis (GraphPad Prism 8.0).

[0364] Cell Culture:

[0365] All cell lines were cultured in media supplemented with 2 mM L-glutamine (v / v), 100 U / l Pen-Strep (v / v) and 10% FBS (v / v) and grown in a humidified environment with 5% CO2 at 37°C. PANC1 (ATCC - CRL- 1469), U2-OS (ATCC HTB-96), HCT116 (ATCC - CCL-247), OVCAR-8 (ATCC), IMR-90 (ATCC - CCL- 186), HEK293 (ATCC - CRL-1573), SW837 (ATCC - CCL-235), SW620 (ATCC - CCL-227), T84 (ATCC - CCL-248) and PATU8902 (DSMZ - ACC179) were cultured in complete DMEM. A549 (ATCC - CCL- 185), NCI-H460 (ATCC - HTB-177), MM415 (Sigma - 10092319) and BxPC3 (CRL-1687) complete RPMI. Panc-08.13 (ATCC - CRL-2551) were cultured in complete RPMI with addition of 10 U / mL of human recombinant insulin (Sigma).

[0366] Immunocytochemistry:

[0367] PANC1 cells were seeded at 0.5 x 105cells per well of a 12-well plate containing a sterilised 0.13- 0.17mm glass coverslip in complete DMEM and incubated overnight. Cells were fixed in 4% paraformaldehyde (Sigma) for 15 minutes at room temperature. Cell membranes were stained with wheat-germ agglutinin (Alexa Fluor 594nm conjugate, ThermoFisher Scientific, W11262) as per manufacturer’s instructions. Cell membranes were then permeabilised with 0.1% Triton X100 (sigma) for 4 minutes at room temperature. Blocking was then carried out for 1 hour at room temperature with 10% donkey serum, 1% BSA in PBS. Both PDE8A (rabbit) and c-RAF (mouse) primary antibodies were then diluted 1 :100 in 5% donkey serum, 1% BSA in PBS and cells incubated overnight at 4°C. Secondary Alexa Fluor antibodies were then simultaneously incubated for 1 hour at room temperature. Cells were washed three times in PBS between each of the above steps. Finally, coverslips were then mounted onto glass slides with Prolong Gold Antifade Mountant with DAPI (ThermoFisher Scientific, P36941). PANC1 cells were imaged using a Zeiss confocal microscope (63X oil immersion objective lens).

[0368] Proximity Ligation Assay:

[0369] In situ analysis of the endogenous c-RAF - PDE8A PPI was confirmed in PANC1 cells via Duolink™ Proximity Ligation Assay (Sigma). In summary, following fixation and membrane permeabilization (see immunocytochemistry protocol), PANC1 cells were blocked for 1 hour at 37°C with Duolink® blocking buffer. Primary antibodies (same as immunocytochemistry) raised in different species (mouse and rabbit) were used to detect proteins of interest (i.e., c-RAF and PDE8A), diluted in Duolink® antibody diluent and incubated overnight at 4°C. PLA probes (oligonucleotide-labelled secondary antibodies; PLUS and MINUS) were then introduced to recognise primary antibodies, where hybridization between oligos occurs if probes are <40nm apart. Complete circularisation of oligos by ligase forms a closed circular DNA template that is subsequently amplified by DNA polymerase, a process known as rolling circle amplification (RCA). Hybridisation of amplified oligos with complementary fluorescently labelled oligos (594 Aex / 624 Aem) allow for localisation of the PLA signal (discrete red spots), indicative of a PPI. Cell nuclei were stained with Prolong Gold Antifade Mountant with DAPI (ThermoFisher Scientific, P36941). PLA signal was detected using a Zeiss confocal microscope with a 63X oil immersion objective. Quantification of identified PLA signals were measured using Image J and analysed on GraphPad Prism 8.0.

[0370] Western Immunoblottinq:

[0371] PANC1 protein lysates were harvested using lysis buffer (25mM Tris, 150mM NaCI, 0.1 mM EDTA, 1 % NP-40, 5% glycerol, pH 7.4) supplemented with protease and phosphatase inhibitors (Roche). Protein samples were diluted in SDS sample buffer (10% SDS, 300mM Tris-HCI, 0.05% bromothymol blue, 10% P-mercaptoethanol) and boiled for 10 minutes at 70°C. Proteins were resolved via SDS-PAGE using 4- 12% Bis-Tris gels (NuPAGE), transferred to nitrocellulose membranes (GE Healthcare) and stained with Ponceau S. Membranes were blocked in Intercept TBS blocking buffer (LI-COR) and incubated overnight in primary antibody at 4°C. Membranes were then incubated in IRDye secondary antibody (1 :10,000, LI- COR) for 1 hour at room temperature and immunoreactive bands visualised using the Odyssey CLx imaging system (LI-COR). Densitometry of immunoreactive bands was carried out using Image J software.

[0372] In-Cell Western Assay:

[0373] PANC1 cells (2x104) were seeded into wells of a clear bottom, black 96 well plate (ThermoFisher Scientific) and allowed to grow in complete DMEM (10% FBS, 2 mM L-glutamine, 100 U / l Pen-Strep) until -70% confluency. Cells were then treated with appropriate concentration of DRx-peptides for 4 hours in 2% FBS containing DMEM. Following treatment, cells were rinsed 3 times in PBS and fixed in 4% PFA (in PBS) for 15 minutes at room temperature. Cells were then permeabilised in 0.1 % Triton X100 for 5 minutes, followed by blocking for 1 hour at room temperature with TBS based Intercept T20 blocking buffer (LI-COR). Wells were then incubated overnight at 4°C in c-RAF pS259 (1 :500) and GAPDH (1 :1000, Abeam) primary antibodies. Cells were then incubated in appropriate IRDye 700nm and 800nm labelled secondary antibodies (1 :800, LI-COR) for 1 hour at room temperature. Antibodies were diluted in TBS based Intercept Antibody Diluent (LI-COR). Cells were washed between all antibody incubation steps 3 times in 1X TBS-T for 5 minutes. Following final wash, protein expression (fluorescence) was measured using an Odyssey CLx instrument.

[0374] In Vitro Scratch - Wound Healing Assay:

[0375] PANC1 cells (3x105) were seeded into wells of a 24 well plate and allowed to grow overnight in DMEM (2% FBS, 2mM L-glutamine, 100 U / l Pen-Strep). Confluent PANC1 monolayer was then manually ‘scratched’ using a sterilised p200 pipette tip. Cells were then washed 2 times with PBS to remove detached cells. Cells were then incubated in DMEM (2% FBS, 2mM L-glutamine, 100U / I Pen-Strep) containing appropriate concentration of PF-04957325, DRx-peptide or vehicle (DMSO) and denuded area was immediately imaged (brightfield) using a Nikon Eclipse TS2 microscope (4X objective). Wound was imaged at 24-hour and 48-hour post-treatment time points. The area of wound was quantified using the Wound Healing Size Tool plugin on Image J software and normalised as % gap closure to respective 0- hour measurement

[0028] .

[0376] In Vitro Cytotoxicity Assay:

[0377] HEK293 and IMR-90 cells (1x104) were seeded in wells of a clear bottom 96-well plate containing complete DMEM (10% FBS, 2mM L-glutamine, 100 U / l Pen-Strep). Cells were allowed to grow overnight before treatment with DRx-170 [1 nM - 10 pM] or Vehicle (DMSO) for 24 Hours. Following treatment duration, cell viability was measured using CellTitre Gio® 2.0 Cell Viability Assay (Promega; G7570) as per manufacturer’s instructions.

[0378] 3D-Spheroid Growth Assay:

[0379] PANC1 cells (2x103) were seeded in wells of a round bottom Nunclon™ Sphera™ 96 well-plate with ultralow attachment coating (ThermoFisher Scientific; #174925), containing 200 pL DMEM (2% FBS, 2mM L- glutamine, 100 U / l Pen-Strep). To encourage spheroid formation, PANC1 cells were centrifuged at 250 x G for 10 minutes at room temperature. PANC1 spheroids were then allowed to grow for 3 days at 37°C, 5% CO2, humidified air. Spheroids were then treated with appropriate concentration of DRx-peptide / Afatinib by first removing 100 pL of media from each well (careful not to disturb spheroid), followed by addition of 100 pL drug at 2X concentration. Treatment regime can be seen in Figure 10. Spheroids were imaged at Day 0, 2, 4, 7 and 10 using a Nikon Eclipse TS2 microscope (4X objective) and spheroid area quantified using Image J software. Finally, to assess overall viability of PANC1 spheroid, endpoint CellTitre-Glo® 3D Cell Viability Assay (Promega; G9681 ) was carried out as per manufacturer’s instructions on Day 10 post-treatment.

[0380] Real-Time Cellular Analysis xCELLiqence Assay:

[0381] Label-free cellular growth of human cancer cell lines were measured using the xCELLigence real-time cellular analysis platform (RTCA, Roche Applied Science) as per manufacturer’s instructions. 96-well E- plates, designed with gold biosensors on the bottom of the plate, were utilised to measure cellular impedance within each well. Cellular impedance measures relative cell growth in real-time (measured as ‘cell index’ or Cl). Cell index increases as cells become adherent, proliferate and / or grow in size (and vice versa). For cellular growth analysis, all human cancer cell lines were seeded at 1 x 104cells per well and allowed to adhere for 4-6 hours (in the case of DRx-100 - DRx-170 assessment) or 18-24 hours (in the case of DRx-170 - DRx-175 assessment). Following this, cells were treated with the appropriate concentration of drug(s) and cell index was monitored every 15 minutes. For adherence analysis, PANC1 cancer cells were seeded at 2 x 104cells per well and immediately treated with appropriate concentration of drug for 8 hours (i.e., cell index curve plateaus; indicative of cellular adherence). Cell index was measured every 5 minutes. DMSO only treated cells were used as a negative treatment control. Unless otherwise stated, all treatments were carried out in respective media containing 2% FBS (low) or 10% FBS (complete), and to a final DMSO concentration of < 1%. Cell index Is normalised to treatment timepoint and the rate of growth (i.e., slope of normalised Cl curve) analysed via linear-regression analyses (GraphPad Prism 8.0).

[0382] Statistical Analysis:

[0383] Data were analysed using an unpaired t test or a one-way ANOVA test with follow up Dunnett’s or Tukey’s multiple comparison analysis. Data represented as MEAN ± SEM from > 3 replicates were determined significant by a p value < 0.05. All statistical analyses were carried out using GraphPad Prism 8.0 software.

[0384] PANC1 Cell-Derived Xenograft Study in NSG Mouse Model: Experimental procedures involving animals and their care were performed by Charles River Oncology Division (Germany) and Discovery Division (United Kingdom). These studies were conducted according to all applicable international, national and local laws and followed the national guidelines for the Care and Use of Laboratory Animals of the Society of Laboratory Animal Science (GV-SOLAS). All animal experiment protocols were approved by the regional council Committee on the Ethics of Animal Experiments. For the pharmacokinetics study, adult CD-1 mice were injected intravenously with DRx-174 (bolus, single 5 mg / kg dose), dissolved in 10% DMSO, 5% Tween 80 in 10 mM citrate buffer (pH 5, 1 mg / mL stock). Relative concentration of DRx-174 was quantified in the plasma at 8 time-points (0.083 - 8 Hrs post-dose) and muscle at 4 time-points (0.5 - 8 Hrs). For the tumour study, NSG (NOD scid gamma) mice (female, 4-6 weeks old) were subcutaneously injected with 5x106PANC1 cells (50:50, PBS - matrigel mix) at the left or right flank. Tumour volume (via digital calipers) and body weight was measured twice per week. Once tumour volume in all mice reached 50 - 150 mm3, mice were randomly allocated into one of two treatment groups (n=9per group) and appropriate treatment regimen was started. Mice in vehicle group were administered orally (PO), q.d., 28 days. DRx-174 treatment group was administered with 40 mg / kg DRx-174 (dissolved in 5% DMSO in D5W) IP, q.d., 28 days, Following the 28-day treatment period, mice were monitored for a further two weeks. Terminal sampling of tumours and blood were taken at day 29 (i.e., 24 hours following last treatment(s), n=3 per group) and day 42 (i.e., study end, n=6 per group), and snap frozen in liquid nitrogen and stored at -80°C for future processing in pharmacodynamic investigations. PANC1 tumours were homogenised (hand-held electric homogeniser) in ice-cold lysis buffer, protein quantified via Bradford assay, and protein expression levels assessed via western immunoblotting analysis (as per above western immunoblotting protocol). Results and Discussion

[0385] Prior to this study, the DP-1 peptide was shown to selectively inhibit the c-RAF - PDE8A PPI , subsequently upregulating inhibitory PKA-specific phosphorylation at serine 259 [14, 29, WO 2020 / 163771]. Targeted disruption not only negatively regulated RAF-associated MAPK signalling but also inhibited cellular proliferation; with an in vivo Drosophila model indicating this mechanism was associated with increased sensitisation to apoptosis. Follow up analysis using the same peptide disruptor, in which the cell-penetrating moiety (stearic acid, C18) was substituted with a proprietary protein-based cell-penetrating moiety (Cell Porter®, Portage Pharmaceuticals Ltd.), further validated the potential therapeutic benefit to disrupting the c-RAF - PDE8A PPI in the context of NRAS driven melanoma; with clear in vitro efficacy and preliminary in vivo pharmacodynamic activity being observed.

[0386] No 3D structure of the region of PDE8A, from which the disruptor peptide was derived (Arg454 - Thr465), has been solved. Predictive PEP-FOLD 3.5 software was therefore utilised to model the peptide’s 3D structure (Figure 1), indicating that the peptide contains two overlapping P-sheet elements, forming a potential [3-hairpin formation. These findings suggest this peptide may be a candidate for head-to-tail cyclisation (or alternative cyclisation approaches that would constrain the peptide into said P-hairpin formation)

[0037] . Further assessment of this peptide using PyMol software highlighted that N-terminal Arg454 and C-terminal Thr465 were ~8.6 Angstroms apart (Figure 1 A). Truncation of Thr465 (an already known non-essential residue

[0014] ); reduced the distance between the N- and C-terminus to ~6.4 Angstroms (Figure 1 B). As a shorter distance between the termini is considered favourable when attempting head-to-tail cyclisation of a peptide, Arg454 - Ser464 was considered over Arg454 - Thr465 as the cyclisation candidate. Consequently, peptides DRx-100, DRx-110, DRx-120 were synthesised, along with a negative control peptide with the known hot spots replaced with alanine (DRx-150) (Table 2). These peptides were synthesised with a C-terminal FITC and their ability to bind c-RAF was assessed utilising a fluorescent-based ligand binding assay.

[0387] Peptide array analysis of the region(s) in which PDE8A binds c-RAF highlighted c-RAF’s kinase domain (Val561 - Lys590) as the primary binding region, with additional accessory binding regions containing all known PKA-specific inhibitory serine residues (serine 43, 233, 259 and 621) also identified (Figure 2). (B) Structural analysis, based on a representative protein structure of human c-RAF kinase domain (c-RAF G323 - R618, PyMOL; PDB - 3OMV) suggests that the primary binding region is located on the external surface of c-RAF’s kinase domain, away from the dimerisation interface and at a site removed from the known active site (not shown). Thus, data suggests PDE8A binds to c-RAF at a distinct site from current RAF inhibitors which target (i) the ATP active site (e.g., Sorafenib) or (ii) the dimerisation interface (e.g., PLX8394). As the primary binding region appears to be within the kinase domain of c-RAF, and as c- RAF’s termini are highly disordered making it challenging to purify full-length c-RAF protein, target engagement assessment of FITC-labelled DRx-peptides were assessed with constitutively active human recombinant c-RAF kinase domain (Ser306 - Phe648, GST-tagged) protein (Figure 3). Co-incubation of 50 pg c-RAF protein with increasing concentrations of FITC-labelled DRx-peptides demonstrated DRx- 100, DRx-110 and DRx-120’s ability to directly bind c-RAF protein (Figure 3A-B). DRx-150 did not bind c- RAF at the concentration range tested (Figure 3A). None of the peptides bound GST protein, indicating specific interaction with the c-RAF-kinase domain protein (Figure 3C). Compared with DRx-100 (Kd = 3.68 ± 0.31 pM), truncated DRx-110 (Kd = 1.79 ± 0.13 pM) and cyclised DRx-120 (Kd = 1.66 ± 0.15 pM) bound c-RAF with a significantly higher affinity (Figure 3D). This indicates that C-terminal truncation of Thr465 unexpectedly improved binding affinity to c-RAF. Importantly, DRx-120 bound c-RAF with a similar (i.e., not-significantly different) affinity to DRx-110 - indicating that cyclisation does not negatively impact target engagement. Admittedly, the apparent Kd is not representative of the true binding affinity as the c-RAF protein sample used was not full-length c-RAF (rational for this discussed above). More, binding signal relies entirely upon the detection of fluorescence emitted from the FITC label conjugated to each peptide. Thus, techniques with higher sensitivity (e.g., surface plasmon resonance, a techniq ue which does not rely upon a fluorescent tag) may demonstrate that DRx peptides possess even higher affinity for c-RAF.

[0388] Conjugating a cell-penetrating moiety to the original disruptor peptide sequence was previously shown to be essential for activity when testing in cellular models, indicating that the native peptide alone is not cell permeable [14,29]. More, these studies highlighted that the choice of cell-penetrating moiety (and hence the cell permeability of the peptides) may influence efficacy. In the present study the D-isomer TAT cellpenetrating peptide was tested (D-TAT). D-TAT (Grkkrrqrrr) is a well characterised cell-penetrating peptide (CPP) that has been evaluated extensively pre-clinically and clinically (e.g. in the phase III clinical candidate molecule AM-111 / XG-102: NCT02561091) [30-32]. D-TAT is not only highly efficient in transporting cargo across cell membranes (including nuclear membrane), but it is also easily synthesised by standard chemical manufacturing processes and demonstrates a good safety profile. As such, D-TAT is considered to have high developability.

[0389] Previous findings have suggested that conjugating a CPP to the C-terminus is a low-risk approach, as this location is removed from the hot spot regions of the peptide (Arg454 / Arg455 and Glu460 / Tyr461)

[0014] . Furthermore, peptide array analysis of the central Gly458 residue within the disruptor peptide indicates that substitution to cysteine does not negatively impact binding and is therefore another appropriate location for conjugating a CPP (Figure 4). Cell-permeable DRx-peptides were synthesised by conjugating D-TAT to the disruptor peptide via a non-cleavable thio-ether (CIAc bridge) linker at the C- terminus or at Gly458Cys (Table 1). Based on findings demonstrating enhanced cell-penetration vs. native TAT CPP, Cyclic TAT (head-to-tail) was also assessed as a potential CPP candidate [33-34]. The activity of these peptides (and the non-cell permeable DRx-peptides) were assessed in cellular in vitro assays. Note that all linear peptides were synthesised with N-terminal acetylation and C-terminal amidation (excluding DP-1 and PPL-008C), an approach intended to further increase peptide stability.

[0390] Peptide array screening analysis indicated that substituting the central Leu456 - Asn459 region with nonnative D-amino acids did not negatively impact binding (Figure 16) and so these positions may be candidates for further modification. Additionally, point substitution of R455, L456, S457, N459, V462 and S464 indicate potential substitutions capable of retaining or increasing binding affinity for c-RAF (Figure 17). To assess the impact of cell-penetrating moieties on the activity of the disruptor peptides, all linear DRx- peptides were tested against a RAS / RAF-driven human pancreatic cancer cell line (PANC1) utilising the real-time cellular analysis platform - xCELLigence (Figure 5). With the exception of DP-1 , all peptide disruptors that contained a cell-penetrating moiety significantly inhibited the normalised cell index (indirect measurement of cellular growth) vs. vehicle and DRx-110 (linear, no cell-penetrating moiety) (Figure 5). Observed previously in MM415 cells (a human NRAS driven melanoma model), PPL-008C again demonstrated superior inhibitory activity vs. DP-1 , suggesting stearic acid is in-effective at delivering the disruptor peptide across the cell membrane (Figure 5)

[0029] . PANC1 growth inhibition was further potentiated when disruptor peptides were conjugated to D-TAT or cyclic TAT (Figure 5). PF-04957325 (PDE8 selective enzymatic inhibitor) markedly inhibited PANC1 cell growth, but to a lesser extent than all TAT CPP conjugated peptides. No significant differences were observed between cyclic TAT or D-TAT. Truncated disruptor peptides (DRx-130, DRx-160 and DRx-190) induced the most potent inhibitory activity of all linear peptides. Finally, conjugating D-TAT at Gly458Cys (DRx-190) did not negatively impact activity (Figure 5). These findings reinforce the need for CPP conjugation and highlight cyclic TAT or D-TAT as the superior candidate vs. Cell Porter® and stearic acid (and vs. PF-04957325). As no significant difference was observed between cyclic TAT and D-TAT, and as D-TAT has been validated more extensively than cyclic TAT, D-TAT was selected as the CPP in which to synthesis to the cyclic disruptor peptide (DRx-170). D-TAT was conjugated via a thioether linker to the cyclic disruptor peptide (DRx-120) at Gly458Cys, creating DRx-170 (Figure 6).

[0391] The RTCA xCELLigence platform was further utilised to evaluate the effect head-to-tail cyclisation and / or conjugation of D-TAT has on DRx-peptide disruptor activity (Figure 7). DRx-120 markedly inhibits PANC1 growth and is dramatically enhanced when conjugated to D-TAT (% nCI inhibition: DRx-120 - 41 .45% vs. DRx-170 - 96.51 %). Findings suggest that cyclic DRx-120 possesses cell-penetrating capability. However, enhancing DRx-120 permeability by conjugating D-TAT (DRx-170) further potentiates activity (Figure 7).

[0392] Finally, to investigate the influence head-to-tail cyclisation has on DRx-peptide disruptor activity vs. a respective linear DRx-peptide disruptor, DRx-170 was assessed against DRx-190 over a longer 60-hour treatment time. Findings clearly demonstrated that DRx-170 and DRx-190 potently inhibit PANC1 growth (Figure 8). However, DRx-170 induced superior inhibitory activity against PANC1 , indicated by a significant difference in the compounds IC50: 0.071 pM (DRx-170) vs. 0.29 pM (DRx-190) (Figure 8). DRx- 170 and DRx-190 were then assessed in high serum (10% FBS) conditions at equimolar concentrations (0.3 pM) to determine if high serum compromised activity (indirectly providing insight into compounds stability) (Figure 9). DRx-170’s activity was similar to that observed in serum reduced (2% FBS) conditions (% nCI: 2% FBS - 28.95 % vs. 10% FBS - 38.46 %) (Figure 9). However, DRx-190’s activity was markedly reduced in high serum conditions (% nCI: 2% FBS - 38.88 % vs. 10% FBS - 68.98 %) (Figure 9). As DRx-170 is cyclised and therefore possesses increased structural rigidity, these findings suggest DRx-170 may be more stable than DRx-190. This notion is reinforced when observing the rate of PANC1 growth (slope of curve), where-by DRx-190 (but not DRx-170) ceases to significantly inhibit the rate of growth 24 hours after initial treatment time point (Figure 9). Thus, DRx-170 demonstrates significantly superior activity vs. DRx-190.

[0393] Proximity ligation assay was carried out to assess the peptides’ ability to penetrate the cell membrane and subsequently disrupt the endogenous c-RAF - PDE8A PPI in PANC1 cells (Figure 10). Cells were treated at equimolar concentrations (1 pM) for only 4 hours to identify which of the DRx-peptides can induce rapid on-target PPI disruption. Notably, only DRx-170 was able to significantly inhibit c-RAF - PDE8A protein complex formation. Next, an in-cell western assay was utilised to investigate the DRx- peptides ability to upregulate a well validated marker of PKA-associated c-RAF inhibition (pS259 c-RAF: a key marker linked with the disruption of the c-RAF - PDE8A PPI

[0014] ) (Figure 11). As with the PLA experiment, PANC1 cells were treated at equimolar concentrations (3 pM) for 4 hours and pS259 c-RAF protein expression was quantified. Again, DRx-170 was the only peptide capable of significantly upregulating pS259 c-RAF expression (Figure 11 A). These findings indicate that combination of the cyclic peptide disruptor with D-TAT CPP significantly improved peptides ability to permeate the cell-membrane and induce rapid on-target activity. Consequently, DRx-170 was selected as the lead candidate peptide disruptor.

[0394] To further determine DRx-170’s ability to induce on-target activity, western immunoblotting was carried out to measure upregulation of pS259 c-RAF protein expression

[0014] , as well as to determine pS43 c-RAF levels (direct marker of RAS - c-RAF PPI displacement) (Figure 11 B-C). DRx-170 markedly upregulates pS259 c-RAF expression, with most significant increase being observed following the 3 pM - 4-hour treatment (Figure 11 B). Additionally, for the first time, significant upregulation of pS43 c-RAF was observed at both 24 hours (3 pM) and 72 Hours (0.3 pM and 3 pM) (Figure 11 C). Data suggests DRx-170 associated c-RAF S259 phosphorylation precedes S43 phosphorylation and indicates c-RAF is being (i) driven into a closed inactive conformation (pS259) and (ii) displaced from upstream activator RAS (pS43). This allosteric mode of c-RAF inhibition does not appear to be driven by its kinase activity as neither S338 c-RAF phosphorylation (precedented marker of c-RAF kinase activity) or downstream pERK1 / 2 levels were influenced. Consequently, DRx-170 is thought to promote c-RAF inhibition independent of its kinase activity in the context of KRAS-driven PDAC. Of note, MAPK signaling pathway is expendable in many RAS-driven malignancies and therefore low basal c-RAF kinase activity may explain why no observable change to pS338 c-RAF or pERK1 / 2 was observed [9]. As previous iteratives have demonstrated an ability to modulate pERK1 / 2 levels (e.g., PPL-008C in an NRAS driven model of malignant melanoma), it is likely that DRx-170 would negatively regulate c-RAF kinase activity in contexts where MAPK signaling is a primary driver of cancer cell growth [14, 29]. In such cases, consideration for combination c-RAF and B-RAF inhibition should be considered as paradoxical activation of MAPK signaling is well documented in B-RAF inhibited cancers harbouring a gain-of-function BRAF mutation (e.g., V600E BRAF driven malignant melanoma) [4, 29].

[0395] As the RTCA xCELLigence analysis platform measures cellular growth via a combination of changes in cell proliferation, size, adhesion, and migration - DRx-170’s ability to inhibit the growth of cancer cells (independent of cellular adhesion, size, or migration) was assessed utilising a PANC1 3D spheroid growth assay (Figure 12). Data demonstrates DRx-170’s ability to (i) slow the rate of spheroid growth (Figure 12) and (ii) significantly inhibit viability of PANC1 spheroid after 10 days of treatment vs. Vehicle (Figure 12). DRx-150 did not significantly inhibit PANC1 spheroid area or viability (Figure 12). Though these findings clearly demonstrate an anti-proliferative role for DRx-170, the level of growth inhibition is not as significant as observed in xCELLigence RTCA experiments. This difference may be due to DRx- 170 influencing additional cellular behaviours such as cell adhesion / migration, which would contribute to the increased potency observed in RTCA assays.

[0396] Following on from single therapy assessment of DRx-170, a combination treatment approach with Afatinib (2ndgeneration EGFR-family tyrosine kinase inhibitor) was assessed. Previous findings in highly aggressive GEM models of PDAC demonstrated convincingly the therapeutic potential of combining c- RAF inhibition with EGFR inhibition [8]. Moreover, PDAC treatment sensitivity to EGFR-family inhibitors is known to correlate with receptor tyrosine kinase enrichment profile (i.e. , high RTK enrichment suggests high sensitivity to EGFR-family inhibitors)

[0036] . Incidentally, as PANC1 cells are RTK enriched - combination with DRx-170 was explored utilising the PANC1 3D spheroid growth assay (Figure 12)

[0036] . As with DRx-170, Afatinib monotherapy was shown to significantly inhibit spheroid growth and viability vs. DRx-150 and Vehicle controls (Figure 12). Combining DRx-170 and Afatinib further potentiated spheroid growth and viability inhibition, with the combination therapy being significantly more efficacious than respective monotherapies (Figure 12). These findings add to the increasing body of evidence that dual c- RAF - EGFR inhibition is synergistic and demonstrates for the first time that DRx-170 - Afatinib is a potentially promising combination strategy.

[0397] Both c-RAF and PDE8A are known to regulate cellular migration, however it remained unclear whether disruption of the c-RAF - PDE8A PPI would influence this. A wound healing scratch assay was therefore utilised to assess the migration (invasion) of PANC1 cells over a 48-hour period following treatment with DRx-170 (Figure 13). Compared with Vehicle, DRx-150 did not significantly inhibit PANC1 cell migration (Figure 14). However, DRx-170 at 0.1 , 1 and 10 pM did significantly inhibit PANC1 migration at both 24- hour and 48-hour time points (Figure 13). These findings indicate that disruption of the c-RAF - PDE8A PPI attenuates PANC1 cells’ ability to migrate and defines an anti-cancer role for DRx-170 beyond inhibition of cancer cell growth.

[0398] Further to migration, RTCA xCELLigence was utilised to assess the effect DRx-170 has on PANC1 cell adherence (Figure 14). DRx-170 potently inhibits the adherence of PANC1 cells, with an IC50 of 46.08 nM. DRx-170 appears to be specific to c-RAF inhibition (as seen by Sorafenib) and not PDE8A (as seen by PF-04957325). Though not assessed, PF-04957325 may have induced inhibition of PANC1 adherence at higher concentrations. Notably, DRx-170 was more potent than both Sorafenib and PF- 04957325, indicating a clear beneficial role of inhibiting the c-RAF - PDE8A complex over the inhibition of individual respective targets. Findings reinforce the multi-faceted anti-cancer activity of targeting the inhibition of the c-RAF - PDE8A PPI with DRx-170, exploiting cancer cell adherence, migration, and proliferation. Finally, to determine the potential therapeutic benefit to disrupting the c-RAF - PDE8A PPI in RAS-RAF driven cancers, DRx-170 [100 nM] was tested against a panel of RAS / RAF mutant human cancer cell lines (Figure 15). Significantly, with the exception of U2-OS - a RAS / RAF wild-type human osteosarcoma cell line, DRx-170 attenuated the cellular growth of all cell lines. Findings highlight that, unlike mutant selective RAS inhibitors (e.g. AMG-510, MRTX1133, etc.) or RAF inhibitors (e.g. Vemurafenib), DRx-170 represents a broad-spectrum inhibitor capable of targeting a wide range of RAS - RAF mutant cancers.

[0399] In vitro ADME (absorption, distribution, metabolism, and excretion) assessment of DRx-170 utilising a 2 hour plasma stability assay highlighted that DRx-170 was stable in rat plasma (> 180 mins), but markedly less so in mouse (20.5 mins) and human (67 mins) (Figure 18). Modifications capable of improving plasma stability were therefore considered.

[0400] DRx-173, DRx-174 and DRx-175 (Table 1) were designed. Briefly, compared to DRx-170, all three of these peptides contain D-Leu, D-Ser, D-Cys and D-Asn instead of the respective L-amino acids in DRx- 170.

[0401] DRx-173 is conjugated to D-TAT CPP in the same manner as DRx-170 and is also cyclised head-to-tail like DRx-170.

[0402] DRx-174 contains a substitution of Val462 to Cys, and is cyclised by a disulphide bond between the side chains of dCys458 and Cys462. The N-terminal Arg454 is acetylated. A further Cys residue (Cys465) was added at the C-terminus, the side chain of which is conjugated to D-TAT via a thio-ether as with DRx-170 and DRx-173.

[0403] DRx-175 also contains a substitution of Val462 to Cys, and the peptide is cyclised (i) via a disulphide bond between the side chains of dCys458 and Cys462, and (ii) head-to-tail, to yield a bicyclic peptide. As bicyclic peptides often possess superior cell-penetrating abilities vs. their respective monocyclic and linear iteratives, and as no rational location for conjugation of a cell-penetrating peptide was available, no cell-penetrating peptide was conjugated to DRx-175.

[0404] All modifications made were chosen based on peptide array data highlighting that Leu456 - Asn459 and Val462 were potentially amenable to modification (Figure 17). ChemDraw structures of DRx-173, DRx- 174 and DRx-175 are shown in Figure 19B-E.

[0405] A control peptide DRx-170-C was also synthesised, containing substitutions of residues believed to be functionally significant (Arg454 to Glu; Arg455 to Glu; Glu460 to Ala; Tyr461 to Ala).

[0406] Before assessment of in vitro activity, DRx-170 - DRx-175 stability in whole blood samples derived from mice was determined (Figure 19A). Peptides were incubated for up to 2 hours. The calculated half-lives were: DRx-170 - 59 mins, DRx-173 - 95 mins, DRx-174 - >250mins, DRx-175 - >250 mins. These results indicate that substituting Leu456 - Asn459 for their respective D-forms enhances in vitro stability in mouse blood. However, unexpectedly, DRx-174 and DRx-175 were markedly more stable, with 100% of compound recovered after 2 Hrs. This indicates that the alternative approach to peptide cyclisation (i.e., D-Cys458 - Cys462, Table 3) results in superior stability characteristics than head-to-tail cyclisation via Arg454 - Ser464 alone.

[0407] As determined by RTCA xCELLigence assessment of DRx-170 - DRx-175 against PANC1 relative cell growth, DRx-173 / 174 / 175 possess similar levels of potency to DRx-170 (i.e., no significant difference in relative growth IC50s) (Figure 20). Not only were all peptides tested at high-FBS (10%) conditions, DRx- 170 - DRx-175 inhibited PANC1 growth significantly more than vehicle and the negative control peptide DRx-170-C at equimolar concentrations. To ensure PANC1 growth inhibition was a consequence of on- target disruption of c-RAF - PDE8A, proximity ligation assay was carried out. PANC1 cells treated with 10 pM DRx-170 / 173 / 174 / 175 (4 Hrs) expressed a significantly lower level of c-RAF - PDE8A complex formation (Figure 21). This was not seen in vehicle or DRx-170-C negative controls. More, PF-04857325 (PDE8 catalytic site inhibitor) and MRTX1133 (KRAS G12D inhibitor) did not negatively influence c-RAF - PDE8A complex formation. Not only do these findings indicate that DRx-173, DRx-174 and DRx-175 are inducing on-target anti-cancer activity vs. PANC1 PDAC cells to a degree comparable to DRx-170, but that the mechanism of cancer growth inhibition (i.e., c-RAF - PDE8A disruption) is differentiated from small molecule inhibitors of KRAS G12D and PDE8 catalytic activity. Worth noting, DRx-175 was less (not significantly) potent than DRx-170 / 174.

[0408] As with DRx-170, DRx-174 significantly inhibited PANC1 cell migration ( / n vitro wound healing assay, Figure 22), as well as the relative cell growth of a panel of human colorectal and pancreatic cancer cell lines harbouring different KRAS mutations (Figure 23). To evaluate the therapeutic potential of combining DRx-174 with clinical candidate drugs targeting the EGFR - RAS signalling pathway, DRx-174 was combined with Afatinib (EGFR-family inhibitor), MRTX1133 (RAS G12D inhibitor), AMG510 (RAS G12C inhibitor) and RMC-6236 (multi RAS(ON) inhibitor). In PANC1 pancreatic cancer cells, which are heterozygous KRAS G12D (and therefore have on KRAS wild-type allele), DRx-174 enhanced anticancer activity of both Afatinib (Figure 24) and MRTX1133 (Figure 25). This was also true for Panc08.13 (KRAS G12D homozygous, Figure 26), SW837 (KRAS G12C homozygous, Figure 27) and HCT116 (KRAS G13D homozygous, Figure 28), where DRx-174 enhanced growth inhibitory activity of MRTX1133, AMG510 and RMC-6236 respectively. These data provide evidence that combining the disruption of c-RAF - PDE8A with EGFR-family inhibitors or RAS-inhibitors can potentiate anti-cancer activity (potency and duration of response), as well as potentially mitigate the risk of acquired resistance observed with these clinical-stage drugs.

[0409] Prior to translating DRx-174 anti-cancer activity in vivo, confirmation that DRx-174 can be detected in the circulation of mice was determined (Figure 29). Plasma and muscle tissue collected from CD-1 mice administered with a single dose of DRx-174 (IV, 5 mg / kg) indicated rapid tissue penetration (<0.5 Hrs) and a relatively short circulating half-life (~0.5 Hrs), with DRx-174 detection up to 4 Hrs post-dosing. As DRx-174 demonstrated a relatively short half-life following IV-administration (Figure 29), dosing frequency in a subsequent tumour in vivo study would need to be high (at least daily), and (as comprehensive dose- formulation of DRx-174 was not yet conducted) alternative routes of administration would need considered to enable higher dosing concentrations (e.g., intraperitoneally).

[0410] Thus, DRx-174 was dosed intraperitoneally at 40 mg / kg daily in mice, over a 28-day period, followed by a 14-day treatment-free period. This was conducted in a sub-cutaneous PANC1 cell-derived xenograft (CDX) immunodeficient (NSG, NOD scid gamma) mouse model, and primary tumour growth was measured and compared with vehicle treatment arm (Figure 30). Treatments began when all tumours reached 50-150 mm3(mean starting tumour volume = 84.03 ± 18.66mm3) and were subsequently normalised to 100% tumour volume (nTV). DRx-174 significantly inhibited tumour growth vs. vehicle by approximately 40% (683.2% ± 79.1% vs. 1081 .4% ± 84.3%, Figure 30). To confirm that tumour growth inhibition was a consequence of on-target activity, protein levels in excised tumours was determined (Figure 31). pS259 c-RAF protein expression was significantly upregulated at day 28 and day 42 in DRx- 174 vs. vehicle, consistent with in vitro data. Interestingly prolonged and repeating dosing of DRx-174 resulted in a significant downregulation of PDE8A1 / 2 and c-RAF protein expression. Finally, DRx-174 induced significant downregulation of pY705 STAT3 and STAT3, adding further evidence that indicates signalling cross-talk between c-RAF and STAT3

[0038] . Thus, DRx-174 elicited on-target anti-tumour activity in a KRAS mutant mouse model.

[0411] Conclusions

[0412] Through a novel mechanism driven by the disruption of the c-RAF - PDE8A PPI, DRx-174 and related molecules represent first-in-class inhibitors of c-RAF with clear anti-cancer activities. DRx-174 demonstrates therapeutic efficacy against a broad spectrum of RAS-RAF mutant human cancer cell lines in vitro, and also shows activity against cell-line derived xenografts in an in vivo model. Importantly, these compounds are markedly differentiated from current RAS / RAF / PDE8 inhibitors and therefore offer a promising approach to treating RAS-RAF driven cancers.

[0413] ***

[0414] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

[0415] While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

[0416] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0417] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0418] References

[0419] A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

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[0432] 13. McCormick, F. (2018) ‘c-Raf in KRas Mutant Cancers: A Moving Target’, Cancer Cell. 33(2):158- 159.

[0433] 14. Brown, K. M. et al. (2013) ‘Phosphodiesterase-8A binds to and regulates Raf-1 kinase’, Proceedings of the National Academy of Sciences, 110(16):E1533-E1542.

[0434] 15. Maurice, D. H. (2013) ‘PDE8A runs interference to limit PKA inhibition of Raf-1’, Proceedings of the National Academy of Sciences of the United States of America. 110(16):6248-6249.

[0435] 16. Cook, S. J. and McCormick, F. (1993) ‘Inhibition by cAMP of Ras-dependent activation of Raf’, Science. 262(5136):1069-1072.

[0436] 17. Wu, J. et al. (1993) ‘Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate’, Science. 262(5136):1065-1069. 18. Mischak, H. et al. (1996) ‘Negative regulation of Raf-1 by phosphorylation of serine 621 ’, Molecular and Cellular Biology. 16(10):5409-5418.

[0437] 19. Sidovar, M. F. et al. (2000) ‘Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase’, Journal of Biological Chemistry, 275(37):28688- 28694.

[0438] 20. Dhillon, A. S. et al. (2002) ‘Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259’, Molecular and Cellular Biology. 22(10):3237-3246.

[0439] 21. Dumaz, N., Light, Y. and Marais, R. (2002) ‘Cyclic AMP Blocks Cell Growth through Raf-1- Dependent and Raf-1 -Independent Mechanisms’, Molecular and Cellular Biology. 22(11 ):3717- 3728.

[0440] 22. Stork, P. J. S. and Schmitt, J. M. (2002) ‘Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation’, Trends in Cell Biology. 12(6):258-266.

[0441] 23. Dumaz, N. and Marais, R. (2003) ‘Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras’, Journal of Biological Chemistry. 278(32):29819- 29823.

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[0445] 27. Amartely, H., Losub-Amir, Anat. And Friedler, Assaf. (2014) ‘Identifying protein-protein interactions sites using peptide arrays’, Journal of Visualized Experiments. (93):52097.

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Claims

Claims1 . A c-RAF inhibitor, comprising a cyclic component P which is capable of binding to c-RAF and inhibiting interaction between c-RAF and PDE8A, wherein said cyclic peptide component is:(i) a cyclic peptide comprising:(a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or an all-D or retro-inverso form thereof; or(b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or a retro-inverso form thereof;(ii) a peptide differing from (i) by no more than 5 substitutions; or(iii) a peptoid or N-methyl version of (i) or (ii); or a pharmaceutically acceptable salt thereof.

2. A c-RAF inhibitor according to claim 1 , wherein P comprises one, two or all three of R1 , E7 and Y8, or a D-form, N-methyl, or N-substituted glycine analogue thereof.

3. A c-RAF inhibitor according to claim 1 or claim 2 wherein P comprises R2 and / or L10 or a D-form, N-methyl, or N-substituted glycine analogue thereof.

4. A c-RAF inhibitor according to any one of the preceding claims wherein cyclisation is via formation of covalent bonds between at least:- an N-terminal functional group of the backbone and a C-terminal functional group of the backbone;- a side chain functional group and an N-terminal functional group of the backbone;- a side chain functional group and a C-terminal functional group of the backbone; or- two side chain functional groups.- N- and C-terminal functional groups and one side chain functional group, via a trifunctional scaffold moiety;- two side chain functional groups and an N- or C-terminal functional group, via a trifunctional scaffold moiety.

5. A c-RAF inhibitor according to any one of the preceding claims further comprising a heterologous moiety.

6. A c-RAF inhibitor according to claim 5 wherein the heterologous moiety is a membrane transit moiety, a pharmacokinetic-modifying moiety, or a proteolysis targeting moiety.

7. A c-RAF inhibitor according to claim 5 wherein the membrane transit moiety is a cell penetrating peptide (CPP).

8. A c-RAF inhibitor according to claim 7 wherein the CPP comprises or consists of GRKKRRQRRR, Grkkrrqrrr, cyclo[GRKKRRQRRR], cyclo[Grkkrrqrrr], C*(Cn)R4* or K*(Cn)R4* e.g. GRKKRRQRRR, Grkkrrqrrr, G*RKKRRQRRR*, Grkkrrqrrr*, C*(CI2)R4* or K*(CI2)R4*.

9. A c-RAF inhibitor according to any one of claims 5 to 7 wherein the heterologous moiety is linked to a terminal backbone functional group of P, or to a side chain of P.

10. A c-RAF inhibitor according to claim 9 wherein the heterologous moiety is linked to a side chain of a residue at position 5, 9 or 11 of P.

11. A c-RAF inhibitor according to claim 10 wherein the residue at position 5, 9 or 11 of P is Ser, Cys, Lys or Gin.

12. A c-RAF inhibitor according to any one of the preceding claims, wherein P has a sequence consisting of:X1 -X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 -X12 whereinXI is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereofX2 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X3 is any residue, e.g. Leu, Nle or an aromatic residue;X4 is any residue, or a residue 4) orX5 is any residue, or a residue 4) orX6 is any residue, or a residue 4> orX7 is Glu or Asp, or a D-form, N-, methyl, or N-substituted glycine analogue thereof;X8 is an aromatic residue, e.g. Tyr, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X9 is any residue, or a residueX10 is Leu or Nle, or a D-form, N-methyl, or N-substituted glycine analogue thereof;XI I is any residue, or a residueX12 is absent or is a residuewherein:P contains a maximum of one residue 4) and one residue ^P; the side chain of any residue 4) forms a covalent bond with the side chain of a residue with the terminal backbone functional group of residue X11 , or with a scaffold moiety; the side chain of any residue ^P forms a covalent bond with the side chain of a residue 4>, with the terminal backbone functional group of residue X1 , or with a scaffold moiety; and the side chain of any residue forms a covalent bond with a heterologous moiety or with a scaffold moiety; and wherein P is:(i) a peptide consisting of:(a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form of either; or(b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4) or a retro-inverso form of either;(ii) a peptide differing from (i) by no more than 5 substitutions; or(iii) a peptoid version of (i) or (ii).

13. A c-RAF inhibitor according to claim 12, wherein:XI is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X2 is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X3 is Leu, Nle, or an aromatic residue (e.g. Leu, Nle, Phe, Tyr, Trp or His), or a D-form, N-methyl, or N- substituted glycine analogue thereof;X4 is Ser, Ala, Gly, Cys, Pro, Gin, Asn, His or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue 4> orX5 is Gly, Ala, Cys, Pro, Gin, Asn, Leu, Nle, His, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue 4> orX6 is Asn, Cys, Met, Gin or His, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue 4> orX7 is Glu or Asp, e.g. Glu, or a D-form, N-, methyl, or N-substituted glycine analogue thereof;X8 is Tyr, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X9 is Vai, Ala, Gly, Cys, Met, Pro, Gin, Asn, Ser, Phe or Trp, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residueX10 is Leu or Nle, or a D-form, N-methyl, or N-substituted glycine analogue thereof;XI I is Ser, Ala, Gly, Met, Pro, Gin or His, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residueX12 is absent or is a residuewherein:P contains a maximum of one residue 4> and one residue ^P;the side chain of any residue 4) forms a covalent bond with the side chain of a residue with the terminal backbone functional group of residue X11 , or with a scaffold moiety; the side chain of any residue ^P forms a covalent bond with the side chain of a residue 4), with the terminal backbone functional group of residue X1 , or with a scaffold moiety; and the side chain of any residue forms a covalent bond with a heterologous moiety or with a scaffold moiety; and wherein P is:(i) a peptide consisting of:(a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form of either; or(b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4), or a retro-inverso form of either;(ii) a peptide differing from (i) by no more than 5 substitutions; or(iii) a peptoid version of (i) or (ii).

14. A c-RAF inhibitor according to claim 12 or claim 13, wherein:XI is Arg, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X2 is Arg, a D-form, N-methyl, or N-substituted glycine analogue thereof;X3 is Leu or Nle, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X4 is Ser, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residueX5 is Gly or an N-methyl analogue thereof, or a residue 4> orX6 is Asn, or a D-form, N-methyl, or N-substituted glycine analogue thereof,X7 is Glu, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X8 is Tyr, or a D-form, N-methyl, or N-substituted glycine analogue thereof;X9 is Vai, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residue ^PX10 is Leu, or a D-form, N-methyl, or N-substituted glycine analogue thereof;XI I is Ser, or a D-form, N-methyl, or N-substituted glycine analogue thereof, or a residueX12 is absent or is a residuewherein:P contains a maximum of one residue 4> and one residue ^P; the side chain of any residue 4> forms a covalent bond with the side chain of a residue ^P, with the terminal backbone functional group of residue X11 , or with a scaffold moiety; the side chain of any residue ^P forms a covalent bond with the side chain of a residue 4>, with the terminal backbone functional group of residue X1 , or with a scaffold moiety; and the side chain of any residue forms a covalent bond with a heterologous moiety or with a scaffold moiety;and wherein P is:(i) a peptide consisting of:(a) the sequence RRLSGNEYVLS (SEQ ID NO: 1) or RRLSGNEYVLS^ (SEQ ID NO: 3), or an all-D or retro-inverso form of either; or(b) the sequence RRIsGnEYVLS (SEQ ID NO: 2) or RRIsGnEYVLS^ (SEQ ID NO: 4), or a retro-inverso form of either;(ii) a peptide differing from (i) by no more than 5 substitutions; or(iii) a peptoid version of (i) or (ii).

15. A c-RAF inhibitor according to any one of claims 12 to 14, wherein P contains one residue 4) and one residue and the side chains of residues 4) and ^P form a covalent bond.

16. A c-RAF inhibitor according to claim 15, wherein residues 4> and ^P are independently selected from Cys and dCys and the side chains of residues 4> and ^P form a disulfide bond.

17. A c-RAF inhibitor according to any one of claims 12 to 16, wherein :XI is Arg;X2 is Arg;X3 is Leu, dLeu or Nle;X4 is Ser or dSer;X5 is Gly or a residueX6 is Asn or dAsn;X7 is Glu;X8 is Tyr;X9 is Vai or a residue ^P;X10 is Leu;XI I is Ser;X12 is absent or is a residue18. A c-RAF inhibitor according to claim 17 whereinX1 is Arg;X2 is Arg;X3 is Leu or dLeu;X4 is Ser or dSer;X5 is a residueX6 is Asn or dAsn;X7 is Glu;X8 is Tyr;X9 is Vai;X10 is Leu;X11 is Ser;X12 is absent; and P is cyclised head-to-tail.

19. A c-RAF inhibitor according to any one of claims 12 to 18 wherein P has a sequence selected from:RRLSGNEYVLS; andRRLS NEYVLS;RRIsGnEYVLS; andRRIsGnEYVLS; wherein the residue G is linked to a heterologous moiety; and wherein P is cyclised head-to-tail.

20. A c-RAF inhibitor according to claim 19, wherein the heterologous moiety is a cell penetrating moiety and / or the residue G is a cysteine residue or a D-cysteine residue.21 . A c-RAF inhibitor according to claim 20, wherein the cell penetrating moiety is a cell penetrating peptide, such as GRKKRRQRRR, Grkkrrqrrr, cyclo[GRKKRRQRRR], cyclo[Grkkrrqrrr], C*(Cn)R4* or K*(Cn)R4*, e.g. GRKKRRQRRR, Grkkrrqrrr, G*RKKRRQRRR*, Grkkrrqrrr*, C*(CI2)R4* or K*(CI2)R4*.

22. A c-RAF inhibitor according to claim 21 , wherein P has the sequence R*RLSCNEYVLS* or R*RlscnEYVLS* and the cell penetrating peptide has the sequence Grkkrrqrrr.

23. A c-RAF inhibitor which is the compound DRx-170 or DRx-173.

24. A c-RAF inhibitor according to any one of claims 12 to 16, wherein:X1 is Arg;X2 is Arg;X3 is Leu or dLeu;X4 is Ser or dSer;X5 is a residue 4);X6 is Asn or dAsn;X7 is Glu;X8 is Tyr;X9 is a residue ^P;X10 is Leu;X11 is Ser;X12 is absent or is a residue25. A c-RAF inhibitor according to claim 24, wherein P has a sequence selected from RRIsdJnEY^PLS and RRIs^nEYMJLS .

26. A c-RAF inhibitor according to claim 24 or claim 25, wherein 4) and ^P are independently selected from cysteine and D-cysteine.

27. A c-RAF inhibitor according to any one of claims 14 to 26, wherein the residue is linked to a heterologous moiety, e.g. a cell penetrating moiety.

28. A c-RAF inhibitor according to claim 27, wherein the cell penetrating moiety is a cell penetrating peptide, such as GRKKRRQRRR, Grkkrrqrrr, cyclo[GRKKRRQRRR], cyclo[Grkkrrqrrr], C*(Cn)R4* or K*(Cn)R4*, e.g. GRKKRRQRRR, Grkkrrqrrr, G*RKKRRQRRR*, Grkkrrqrrr*, C*(CI2)R4* or K*(CI2)R4*.

29. A c-RAF inhibitor according to claim 28, wherein P has the sequence RRIsc*nEYC*LSC and the cell penetrating peptide has the sequence Grkkrrqrrr.

30. A c-RAF inhibitor according to claim 29, which is the compound DRx-174.31 . A c-RAF inhibitor according to claim 25 wherein the sequence RRIs^nEY^PLS is additionally cyclised head-to-tail.

32. A c-RAF inhibitor according to claim 31 , wherein P has the sequence R**Rlsc*nEYC*LS**, where ** indicates head-to-tail cyclisation and * indicates disulfide cyclisation between the side chains of the D- cysteine and cysteine residues.

33. A c-RAF inhibitor according to claim 32, which is the compound DRx-175.

34. A c-RAF inhibitor according to any one of the preceding claims for use in a method of medical treatment.

35. A c-RAF inhibitor according to any one of claims 1 to 33 for use in the treatment of a condition characterised by inappropriate activation of the RAS pathway, or susceptible to treatment by inhibition of c-RAF, e.g. a condition characterised by a mutation in a RAS pathway component.

36. A c-RAF inhibitor for use according to claim 35, wherein the condition is a cancer.

37. A c-RAF inhibitor for use according to claim 36, wherein the cancer carries a gain-of-function mutations in Ras (e.g. KRAS, NRAS or HRAS).

38. A c-RAF inhibitor for use according to claim 36 or claim 37 wherein the cancer is: a cancer of the central nervous system (CNS), such as glioblastoma multiforme (GBM) or lower grade glioma (LGG); a cancer of the head and neck, such as head and neck squamous cell carcinoma (HNSC); an endocrine cancers, such as papillary thyroid carcinoma (THCA), anaplastic thyroid carcinoma (THCAA) or follicular thyroid carcinoma (THCAF); a thoracic cancer, such as lung adenocarcinoma (LUAD) or lung squamous cell carcinoma (LUSC); a breast cancer, such as breast invasive carcinoma (BRCA); a core gastrointestinal (Gl) cancer, such as esophageal carcinoma (ESCA), stomach adenocarcinoma (STAD), small intestine adenocarcinoma (SIAD), colon adenocarcinoma (COAD) or rectal adenocarcinoma (READ); an accessory gastrointestinal cancer, such as liver hepatocellular carcinoma (LIHC), cholangiocarcinoma (CHOL), gallbladder carcinoma (GBC) or pancreatic adenocarcinoma (PAAD); a genito-urinary (GU) cancer, such as kidney renal clear cell carcinoma (KIRC), bladder urothelial carcinoma (BLCA) or prostate adenocarcinoma (PRAD); a gynaecological cancer, such as ovarian serous cystadenocarcinoma, uterine corpus endometrial carcinoma (UCEC), cervical squamous carcinoma or endocervical adenocarcinoma (CESC); a skin cancer such as skin cutaneous melanoma (SKCM); or a haematological cancer, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) or plasma cell myeloma (PCM).

39. A c-RAF inhibitor for use according to claim 36, wherein the cancer carries a B-Raf mutation, e.g. wherein the cancer is resistant to treatment with a B-Raf inhibitor.

40. A c-RAF inhibitor for use according to claim 36, wherein the cancer carries a B-Raf mutation, e.g. wherein the cancer is resistant to treatment with a B-Raf inhibitor.41 . A c-RAF inhibitor for use according to claim 36 or claim 39, wherein the cancer is a melanoma, e.g. a B-Raf inhibitor-resistant melanoma.

42. A c-RAF inhibitor according to claim 35, wherein the condition is an inflammatory condition.

43. A c-RAF inhibitor for use according to claim 42 wherein the inflammatory condition is an autoimmune disease, e.g. rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, microscopic colitis, diversion colitis or Behcet’s disease.

44. A c-RAF inhibitor for use according to claim 35, wherein the condition is a RASopathy,45. A c-RAF inhibitor for use according to claim 44, wherein the condition is neurofibromatosis type 1 (NF1), Noonan syndrome (NS), Noonan syndrome with multiple lentigines (NS-ML), Noonan syndrome with loose anagen hair (NS-LH), cardiofaciocutaneous syndrome (CFC), Costello syndrome (CS), Legius syndrome (LS), central conducting lymphatic anomalies syndrome (CCLA), SYNGAP1 syndrome or capillary malformation arteriovenous malformation syndrome (CM-AVM).

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