Artificial peptide having pkmt-inhibitory effect

Abstract

The invention relates to an artificial peptide having a length in the range of 10 to 20 amino acids with an amino acid sequence derived from a section of a substrate for a protein lysine methyltransferase, the peptide having a strong binding affinity to protein lysine methyltransferase, the amino acid sequence allowing a loop structure to be formed, and the peptide having inhibitory effect on the enzymatic activity of the protein lysine methyltransferase.

Description

[0001] Artificial peptide with PKMT inhibitory effect

[0002] The invention relates to an artificial peptide with a strong binding affinity to a protein lysine methyltransferase, a method for producing the peptide and uses of the peptide.

[0003] Human genetic information is stored in the nucleus of every cell in the form of chromatin, a complex of DNA with histone proteins. Which genes are switched on or off is determined by the organization of the chromatin. Protein lysine methyltransferases (PKMTs) are enzymes that act in the nucleus and significantly influence the structure and organization of chromatin [Boriack-Sjodin & Swinger, 2015]. To accomplish this, PKMTs bind to regions of histone proteins (also called histone peptides) and transfer one or more methyl groups to lysine residues within these structures. These lysine methylations often occur in the unstructured N-terminal ends of the histone proteins. The interaction between the PKMT and the histone peptides is of fundamental importance.Different PKMTs insert methyl groups at different sites on different histone peptides, and a specific interaction pattern ensures that exactly the right binding site is found and methylated.

[0004] The incorporation of the methyl group by PKMTs is accomplished with the help of a cofactor that provides an activated methyl group. This cofactor is called S-adenosyl-L-methionine (SAM) and binds together with the histone peptide in the active site of the PKMT (Fig. 1) [Boriack-Sjodin & Swinger, 2015]. The transferred methyl group then acts as a signal and initiates a cascade that leads to the restructuring of chromatin [Ailis & Jenuwein, 2016]. This restructuring determines which genes are switched on and which are not [Ailis & Jenuwein, 2016]. Thus, PKMTs play a central role in gene regulation.

[0005] Defective methylation of histones and other proteins can have far-reaching consequences. Mutations in the PKMT, for example, lead to PKMT hyperactivity and thus to an excess of transferred methyl groups [Kudithipudi & Jeltsch, 2014]. This overmethylation leads to impaired gene regulation, which can manifest itself in the development of various forms of cancer [Kudithipudi & Jeltsch, 2014]. Likewise, dysregulation or overexpression of PKMT can also lead to defective histone methylation patterns. Preventing PKMT hyperactivity is therefore a promising approach for many cancer patients and is already being tested in clinical trials [Copeland, 2018].

[0006] Many conventional approaches to inhibiting PKMTs attempt to disrupt the interaction between PKMT and its cofactor SAM [Schapira, 2016; Copeland, 2018]. This is achieved using inhibitors designed to bind to PKMT instead of SAM (so-called competitive inhibitors, here competitive for SAM). Binding of the inhibitor blocks the enzyme, preventing the transfer of a methyl group.

[0007] However, the use of SAM inhibitors for PKMTs is problematic with regard to the specificity of the molecules, because other methyltransferase enzyme families, such as DNA methyltransferases, RNA methyltransferases, or protein arginine methyltransferases, also use SAM as a donor for methyl groups. SAM derivatives can therefore also interact with them and influence their activity. Furthermore, in defined forms of cancer, specific PKMTs are always altered in their activity and these must be specifically regulated [Kudithipudi & Jeltsch, 2014; Copeland et al. 2013]. Specific PKMT inhibitors would also be valuable reagents in research, allowing the biological function of a specific PKMT to be studied in more detail.

[0008] There is therefore an urgent need to address inhibitors that specifically inhibit certain PKMTs.

[0009] This object is achieved by a method according to claim 1, an artificial

[0010] A peptide according to claim 8, a use according to claim 14, and a use according to claim 15 are solved. Further advantageous embodiments and refinements of the invention emerge from the subclaims, the figures, and the exemplary embodiments. The embodiments of the invention can be advantageously combined.

[0011] A first aspect of the invention relates to a method for producing an artificial peptide having a length in the range of 10 to 20 amino acids with an amino acid sequence derived from a portion of a substrate for a protein lysine methyltransferase (PKMT), wherein the peptide has a strong binding affinity to a PKMT, comprising the steps:

[0012] - Identifying a PKMT peptide substrate with maximum methylatability in a first design process S1 , wherein

[0013] - in a first sub-step S1.1, a peptide array comprising first peptides having a length of 10 to 20 amino acids with amino acid sequences derived from a section of the substrate for a PKMT is provided, wherein the first peptides have different amino acid sequence variants and each have at least one basic amino acid to be methylated, and methylating the peptides by a PKMT,

[0014] - in a second sub-step S1.2, at least one first peptide is identified which has a particularly strong methylatability compared to known substrate peptides of a specific protein lysine methyltransferase,

[0015] - in a third sub-step S1.3, design and production of derived peptides whose amino acid sequence should enable the formation of a loop structure and a basic amino acid to be methylated should be positioned in the center of the loop structure,

[0016] - in a fourth sub-step S1 .4 testing the methylability of the derived peptides,

[0017] - Optimizing the binding of the artificial peptides to the PKMT in a second design process S2, wherein - in a first sub-step 2.1, producing derived peptides from S1, the amino acid sequence of which should enable the formation of a loop structure, wherein a basic amino acid to be methylated should be positioned in the center of the loop structure, and identifying at least one peptide which has a particularly strong binding compared to known substrate peptides of a specific protein lysine methyltransferase,

[0018] - in a second sub-step 2.2, design and production of derived peptides based on the data obtained in the previous design process, the amino acid sequence of which should enable the formation of a loop structure, with a basic amino acid to be methylated being positioned in the center of the loop structure,

[0019] - in a third sub-step S2.3, testing the binding activity of the derived peptides, whereby steps S2.2 and S2.3 are repeated cyclically if necessary,

[0020] - Optimizing the inhibition of PKMT by the artificial peptides in a third design process S3, where

[0021] - on the basis of the data obtained in the previous design process, derived peptides are produced, the amino acid sequence of which should enable the formation of a loop structure, with a basic amino acid to be methylated being positioned at the centre of the loop structure, and then the inhibition of PKMT by the peptides is investigated in order to identify at least one peptide which has a particularly strong inhibition of PKMT, whereby the third design process can be repeated if necessary.

[0022] The steps are also referred to as the design process, which is divided into substeps. In the first design process, artificial substrate peptides that are methylated by the PKMT at the highest possible rate are identified over several cycles. In the next design process, artificial substrate peptides that bind to the PKMT with the highest possible affinity are identified over several cycles. In the third design process, peptides that inhibit the PKMT with the highest possible efficiency are identified from these.

[0023] The peptides from the second design process are produced with a length in the range of 10 to 20 amino acids, based on the amino acid sequence of the identified substrate peptide. The amino acid sequence of the derived peptides should allow the formation of a loop structure, and a basic amino acid to be methylated should be positioned at the center of the loop structure. Subsequently, the inhibition of PKMT by these peptides is investigated to identify at least one peptide with particularly strong inhibition.

[0024] In other words, the process begins with initial peptides, from which the derived peptides are derived, and from which a new peptide is identified. This process can be repeated multiple times, gradually optimizing the methylation, binding, and inhibitory activity of the derived peptides. The final, best-interacting and inhibitory peptides are identical to the artificial peptide of the invention.

[0025] It has surprisingly been found that the method according to the invention can be used to identify at least one peptide that inhibits the activity of a PKMT.

[0026] The term "loop structure" refers to a conformation of a protein, or in the context of the invention, a peptide, that, in the biochemical sense of the lock-and-key principle, fits particularly well with the enzyme's binding pocket. The loop structure is, in particular, a structural element in which a refolding loop ("loop region") is stabilized by interactions between the amino acids upstream and downstream of the loop region ("stem region"). Positioning the basic amino acid to be methylated in the center of the loop structure means that the basic amino acid to be methylated is positioned in the loop region. An advantageous form of a loop structure refers to a U-shaped structure of the peptide with a short end-to-end distance. This creates a hairpin-like loop of the peptide. Other conformations are also possible.

[0027] In a preferred embodiment of the method, a histone protein is chosen as the substrate for a PKMT.

[0028] Preferably, said basic amino acid is a proteinogenic, non-proteinogenic or modified amino acid.

[0029] In a preferred embodiment of the process, the basic amino acid is lysine.

[0030] In a preferred embodiment of the method, the strong binding affinity of the peptide to a PKMT exceeds the binding affinity of a natural histone protein.

[0031] The terms "strong" and "extra strong" in relation to the binding of peptides to a PKMT, which can be illustrated by a degree of methylation, should be viewed relative to other peptides. Strong binding is several times higher than that of conventional peptides. Very strong binding, in turn, is several times higher than that of strongly binding peptides.

[0032] In this sense, a preferred embodiment of the method is advantageous in which a PKMT is selected whose enzyme activity correlates with a disease. This enables the design of a peptide suitable for treating a corresponding disease.

[0033] It is particularly preferred if a PKMT is selected in the method whose enzyme activity correlates with a cancer. This advantageously provides a peptide suitable for treating a cancer.

[0034] It is particularly preferred if a PKMT from a group comprising NSD2, SETD2, EZH2, and DOT1 L is selected in the method. These PKMTs have been shown to correlate with the development of certain types of cancer. It is therefore particularly advantageous if a peptide for inhibiting these PKMTs is provided that is suitable for treating corresponding types of cancer.

[0035] A second aspect of the invention relates to an artificial peptide produced by the method according to the invention, having a length in the range of 10 to 20 amino acids and an amino acid sequence derived from a portion of a substrate for a protein lysine methyltransferase (PKMT), wherein the peptide has a strong binding affinity to a PKMT. The specific amino acid sequence enables the formation of a loop structure. A basic amino acid to be methylated is positioned at the center of the loop structure. The peptide has an inhibitory effect on the enzyme activity of protein lysine methyltransferase.

[0036] The peptide according to the invention is advantageous because it binds a specific PKMT with high specificity, thereby reducing the activity of the PKMT. Initial binding occurs in a loop conformation, in which the peptide according to the invention can bind to the PKMT more quickly and efficiently. Final binding in the active center of the PKMT can occur in a loop conformation or, after unfolding, in an extended form. The inhibitory effect of the peptides occurs through competitive inhibition of the binding of the histone protein to the protein lysine methyltransferase. This selectively prevents the binding of histones (i.e., the true substrates) to the corresponding PKMT. In this context, the peptide according to the invention is advantageously specific compared to conventional approaches because it does not target the SAM binding site of the enzyme and therefore does not inhibit other methyltransferases or other PKMTs.Furthermore, the peptide according to the invention can be used to investigate the biological function of a specific PKMT.

[0037] Surprisingly, it has been found that a peptide with a loop structure is methylated more efficiently by PKMT than the corresponding linear peptide and associates with the PKMT more quickly and efficiently. It is therefore advantageous to incorporate a loop conformation into the design of the peptide of the invention. Further work has shown that the formation of a loop structure enables faster binding to a desired PKMT (for illustration, see Figure 6).

[0038] The loop structure can be held together, in particular, by a chemical bond selected from the group consisting of covalent bonds, hydrogen bonds, or hydrophobic interactions. A covalent bond that is stable and can be achieved via a reaction of functional groups that can be incorporated during the chemical synthesis of the peptides is particularly advantageous. For example, the formation of a disulfide bridge via two cysteine ​​residues is suitable. Therefore, embodiments of the peptide according to the invention whose amino acid sequence contains at least two amino acids that can form a disulfide bridge or another chemical bond that stabilizes the loop structure are advantageous.

[0039] Preferably, the substrate for a PKMT is a histone protein.

[0040] Preferably, the basic amino acid in question is a proteinogenic, non-proteinogenic, or modified proteinogenic amino acid. This allows an amino acid to be selected from a wide biochemical spectrum. Proteinogenic amino acids are those that occur naturally in proteins. Non-proteinogenic amino acids (i.e., acids with an amino group at the Ca atom) are those that occur naturally but are not building blocks of proteins. Modified amino acids are those that exhibit an artificial or natural chemical modification.

[0041] Lysine, arginine, or histidine are suitable proteinogenic basic amino acids. Non-proteinogenic basic amino acids include ornithine or meta-aminophenylalanine. Modified proteinogenic basic amino acids include methylated lysine, methylated arginine, or methylated histidine. Lysine is preferably the basic amino acid. However, other basic amino acids are also possible and are also preferred, especially ornithine.

[0042] Preferably, the peptide is specific for binding to a specific PKMT. The final binding can occur in a looped or extended form. In particular, the peptide is specific for SETD2 (SET-domain containing protein 2, also known as KMT3A). It has been shown that this enzyme interacts particularly efficiently with corresponding peptides. A correlation between hyperactivity of PKMTs such as NSD2, DOT1 L, and EZH and cancer development has been demonstrated. Using an analogous approach, peptides can be developed that are specific for NSD2, EZH2, and DOT1 L, for example.

[0043] Preferably, the specific protein lysine methyltransferase is correlated with a disease with respect to its enzyme activity. It can be assumed that many diseases are correlated with PKMT hyperactivity. In particular, the specific PKMT is correlated with cancer, neurological disorders, brain diseases, inflammatory disorders, metabolic diseases, and diseases of the cardiovascular system. A further advantage of the peptide according to the invention is that, due to the high specificity for a particular PKMT, a therapy can be tailored to a specific disease type, in particular a specific tumor type, and even a specific patient. This enables, for example, personalized cancer therapy, which could give the invention a place alongside already established methods such as antibody or CAR-T cell therapy.

[0044] Preferably, the strong binding affinity of the peptide to a PKMT exceeds the binding affinity of a natural histone protein.

[0045] Preferably, the amino acid sequence of the peptide according to the invention is derived from a known PKMT substrate, for example, in the case of SETD2, from the N-terminal tail region of the histone protein H3. Binding of PKMTs to histone proteins occurs through binding of the enzyme to the freely accessible, so-called "histone tail." This tail represents the N-terminal end of the histone protein, consists of a specific sequence of approximately 20 to 40 amino acids, and contains characteristic lysine residues at certain positions. However, other residues within the histone proteins (e.g., H3K79) and also in non-histone proteins are also methylated by PKMTs. These methylation events also have important biological functions and are therefore potential targets for PKMT inhibitors.

[0046] A third aspect of the invention relates to the use of a peptide according to the invention for inhibiting the enzyme activity of a specific protein lysine methyltransferase. Examples of suitable PKMTs are NSD2, SETD2, EZH2, and DOT1 L. This use relates in particular to a medical use for treating the diseases mentioned below, especially cancer.

[0047] A fourth aspect of the invention relates to the use of a peptide for inhibiting the enzyme activity of a specific protein lysine methyltransferase, wherein a peptide obtained in step S1, S2, or S3 of the method according to the invention is used. All peptides obtained and tested in the design process in step S1, S2, or S3 of the method according to the invention are possible. This includes not only the final peptide obtained, but also the peptides obtained in the intermediate steps. The latter are referred to as substrates or, if they already bind the PKMT very effectively, as supersubstrates. These peptides can also be used as inhibitors of a PKMT. On the other hand, substrate peptides for which a strong inhibitory effect can already be demonstrated are advantageous for use in the further design process to generate even more effective inhibitors.

[0048] For the use of the peptide according to the invention, in particular for medical use, a pharmaceutical composition is advantageous which comprises at least one peptide according to the invention with at least one pharmaceutically acceptable carrier substance, mesoporous nanoparticles, a cryoprotectant, a lyoprotectant, an excipient and / or a diluent.

[0049] A further aspect of the invention relates to a peptide according to the invention for use as a medicament. A medicament against any disease is possible in which the peptide is effective as an inhibitor of a PKMT involved in the cause of the disease. The medicament can be used particularly advantageously in cancer therapy, since treating cancer by inhibiting PKMT using other strategies is already known. However, a medicament that can be used in the treatment of neurological disorders, brain diseases, inflammatory disorders, metabolic diseases, and diseases of the cardiovascular system is also possible.

[0050] A further aspect of the invention relates to a peptide according to the invention for use in the treatment of cancer, in particular of a cancer disease in which defective methylation of histone proteins by a protein lysine methyltransferase occurs.

[0051] The peptide according to the invention is preferably used for the treatment of a cancer of the liver, a cancer of the pancreas, a cancer of the prostate, breast cancer, another solid tumor or a cancer of the hematopoietic system.

[0052] To use a peptide according to the invention, it must be provided in a suitable form. The peptide is used to prepare a formulation for oral, intravenous, topical, intranasal, intraperitoneal, and / or subcutaneous administration and / or inhalation and / or another injectable form.

[0053] Also disclosed is a method for producing a peptide according to the invention by means of solid-phase peptide synthesis.

[0054] Also disclosed is a method for the prophylaxis and / or treatment of cancer in a subject, wherein a peptide according to the invention or a corresponding pharmaceutical composition is administered to the subject. The subject is a mammal, and in a particular embodiment, a human.

[0055] The invention is explained in more detail with reference to the figures.

[0056] Figure 1 shows a schematic representation of the transfer of a methyl group from SAM to the N-terminal end of a histone protein by PKMT.

[0057] Figure 2 is a schematic representation of a peptide according to the invention bound to a PKMT which prevents the docking of the N-terminal end of a histone protein.

[0058] Figure 3 is a flow diagram of an embodiment of a process according to the invention.

[0059] Figure 4 shows an autoradiogram of a spot array for identifying a peptide according to the invention in design cycles.

[0060] Figure 5 shows an autoradiogram of a spot array to demonstrate the specificity of substrates for certain PKMTs.

[0061] Figure 6 Data from a FRET experiment demonstrating a loop conformation adopted by the peptide of the invention in solution.

[0062] Figure ? Results of a controlled molecular dynamics simulation demonstrating that a peptide according to the invention binds more rapidly to a PKMT due to its loop conformation.

[0063] Figure 8 shows an autoradiogram demonstrating the inhibitory effect of certain peptides on a PKMT.

[0064] Figure 1 schematically depicts the methylation of a histone protein 1. The methyl group 3 to be transferred is provided by a cofactor called S-adenosyl-L-methionine (SAM) 4. SAM 4 is bound together with the histone protein 1 in the active site of PKMT 2. There, the methyl group 3 is transferred to a region of the histone protein 1. The transferred methyl group 3 acts as a signal and initiates a cascade that leads to a restructuring of the chromatin, in which genomic DNA and histones are arranged. This restructuring determines which genes can be switched on and which cannot.

[0065] Figure 2 schematically illustrates how, according to the invention, the binding of the N-terminal end of histone protein 1 to PKMT 2 is counteracted. A protein lysine methyltransferase (PKMT) 2 has an active site for the methylation of regions of histone proteins, in which the cofactor SAM 4 and the peptide 10c to be methylated bind. An artificial peptide 10 is provided, which is present in solution in a loop 10b or extended conformation 10a. The artificial peptide associates with a specific PKMT preferably in a loop conformation 10b and ultimately binds in the region of the active site of the PKMT in a loop or extended form 10c. The bound peptide 10c binds more strongly than histone protein 1 and thereby prevents its binding to PKMT 2. Thus, the histone protein cannot be methylated.

[0066] In one embodiment of a process according to the flow diagram of Fig. 3 for producing a peptide according to the invention, the process is divided into three design processes.

[0067] In a first design process S1, artificial PKMT peptide substrates with maximum methylability are identified. In a first substep S1.1, a peptide array containing first peptides with amino acid sequences derived from a segment of a histone protein is provided. The first peptides exhibit various amino acid sequence variants. In a second substep S1.2, at least one first peptide is identified as a substrate that exhibits particularly strong methylability compared to known substrate peptides of a specific protein lysine methyltransferase. Based on these data, further derived peptides are designed and produced in a third substep S1.3. These derived peptides have a length of 10 to 20 amino acids.It is essential that the amino acid sequence of the derived, new peptides should enable the formation of a loop structure and that a basic amino acid to be methylated should be positioned in the center of the loop structure. The loop structure is, in particular, a structural element in which a refolding loop ("loop region") is stabilized by interactions between the amino acids upstream and downstream of the loop region ("stem region"). Positioning the basic amino acid to be methylated in the center of the loop structure means that the basic amino acid to be methylated is positioned in the loop region. In this exemplary embodiment, lysine is chosen as the basic amino acid. In a fourth sub-step S1.4, the methylatability of these peptides is tested. Steps S1.3 and S1.4 are repeated several times if necessary.

[0068] In a second design process (S2), the binding of the artificial peptides to the PKMT is optimized. For this purpose, the previously optimized peptides are produced in a first substep (S2.1). These peptides have a length in the range of 10 to 20 amino acids and are based on the amino acid sequence of the identified substrate peptide. The amino acid sequence of the derived peptides should enable the formation of a loop structure, with a lysine to be methylated positioned at the center of the loop structure. Subsequently, at least one peptide is identified that exhibits particularly strong binding compared to known substrate peptides of a specific protein lysine methyltransferase.

[0069] Based on these data, further derived peptides are designed and produced in a second substep (S2.2). These derived peptides are 10 to 20 amino acids long. It is essential that the amino acid sequence of the derived, new peptides allows for the formation of a loop structure, and that a lysine to be methylated is positioned at the center of the loop structure. In a third substep (S2.3), the binding of these peptides to the PKMT is tested. These steps (S2.2 and S2.3) are repeated several times if necessary (indicated by dashed lines).

[0070] In a third design process (S3), the inhibition of PKMT by the artificial peptides is optimized. In S2, optimized peptides with a length ranging from 10 to 20 amino acids are designed and produced. These peptides are based on the amino acid sequence of the identified substrate peptide. The amino acid sequence of the derived peptides should enable the formation of a loop structure. Subsequently, the inhibition of PKMT by these peptides is investigated to identify at least one peptide with particularly strong inhibition. This step can also be repeated in cycles if necessary (indicated by dashed lines).

[0071] Figure 4 shows a SPOT array on which various peptide sequences derived from the histone 3 protein sequence were synthesized. These were subsequently methylated with the NSD2 PKMT using radiolabeled SAM (Figure 4a). Different positions show a stronger methylation signal due to the individual amino acid changes. Based on this, peptides identified by the initial array were synthesized again on a SPOT array (Experiment (2)). The newly discovered peptides (Tables 1 and 2) show a further increased methylation signal. For example, the peptides in A16, B10, and B13 show a stronger methylation signal than H3K36, the natural substrate of NSD2 (A1 and B15) (Figure 4b). In Experiment 3, the peptide in A8 is identified as the most heavily methylated (Figure 4c). The methylatability of these peptides can be further improved in subsequent design cycles.In the following design processes 2 and 3, the binding of the artificial peptides 10 to NSD2 is optimized, possibly also in several cycles, and the most inhibitory peptides are finally identified. Table 1: Peptide sequences in experiment (2). Table 2: Peptide sequences in Experiment (3)

[0072] Figure 5 shows the results of methylation assays used to demonstrate the specificity of peptides obtained during the design process. The autoradiograms on the left show methylation with the PKMT NSD2. The autoradiograms on the left show methylation with the PKMT SETD2. The exposure times are indicated below the autoradiograms. It can be seen that peptide B1 is particularly effectively methylated by NSD2, and peptide B3 by SETD2. The peptide assignment can be found in the adjacent table.

[0073] Figure 6 shows data from a FRET experiment with peptides in solution, demonstrating that a peptide of the invention adopts a loop structure in solution. The example shows data for the histone peptide (H3K36) and a peptide of the invention (ssK36) that preferentially binds to the PKMT SETD2.

[0074] A) Schematic of the FRET system. The H3K36 and ssK36 peptides were synthesized with an EDANS fluorophore at the C-terminus and a Dabcyl quencher at the N-terminus. EDANS was excited at 340 nm, and the fluorescence emission was measured at 490 nm. Due to FRET, the fluorescence emission is partially quenched by Dabcyl, and only the remaining fluorescence was measured. The FRET experiments were performed at several temperatures, starting at 5 °C up to 95 °C, using peptide concentrations of 10 pM. B) At 5 °C, EDANS-ssK36-Dabcyl showed approximately 33% lower fluorescence intensity than EDANS-ssK36-Dabcyl. At 95 °C, both peptides showed the same intensity. This indicates the preferred loop structure of the peptide ssK36 according to the invention, which is abolished at high temperatures by the enhanced thermal motion.The average fluorescence intensities of Dabcyl-ssK36-EDANS were obtained from three replicates and normalized to 95°C and Dabcyl-H3K36-EDANS. C) A control experiment showed no difference in the fluorescence intensity of EDANS-H3K36-Dabcyl and EDANS-ssK36-Dabcyl after their digestion with proteinase K.

[0075] H3K36: APATGGVKKPHRYRP ssK36: APRFGGVKRPNRYRP

[0076] Figure 7 shows the results of steered molecular dynamics (sMD) simulations, which demonstrate that a peptide of the invention binds more rapidly to a PKMT due to its loop structure. The final binding can occur at various sites within the PKMT enzyme in a loop or extended conformation.

[0077] The sMD of the binding of H3K36 and ssK36 to the SETD2 PKMT was determined with a distance-dependent external force of 0.5 kJ / (mol ■ A 2) between the Ns atom of K36 and the methyl group C atom of SAM bound in SETD2. The number of successful docking of ssK36 to the active center of SETD2 after 50 ns sMD was measured. Criteria used to define a successful docking event are derived from the geometry of the SN2 transition state of methyl group transfer, which is known to those skilled in the art. The sMD simulations were carried out in the presence and absence of an additional, distance-dependent repulsive force of 0.3 kJ / (mol ■ A 2 ) between the peptide ends, which prevents the formation of a loop structure. The figure shows the number of successful docking events in 100 sMD simulations with H3K36 or ssK36 based on the SN2 transition state criteria.

[0078] Figure 8 shows an autoradiogram of a methylation assay used to biochemically demonstrate the inhibitory effect of the peptides obtained in the described design process. Specifically, the inhibition of the PKMT SETD2 was investigated. The methylation of a histone protein analogue with the characteristic N-terminal tail region of the histone protein H3 is shown. The methylation is weakened by the addition of the designed peptide (ssK36), demonstrating the inhibitory effect of the peptide (left side of the image). In a control experiment using a peptide derived from the start sequence of the design process from a histone protein H3 (H3K36), it was shown that a conventional peptide exhibits no measurable inhibitory effect on the PKMT (right side of the image).

[0079] H3K36: APATGGVKKPHRYRP ssK36: APRFGGVKRPNRYRP

[0080] List of reference symbols

[0081] 1 histone

[0082] 2 PKMT

[0083] 3 Methyl group 4 SAM

[0084] 10 artificial peptide

[0085] 10a artificial peptide in solution in stretched structure

[0086] 10b Artificial peptide in solution in loop structure

[0087] 10c Artificial peptide bound to PKMT (the binding can be stretched or in a loop conformation)

Claims

A method for producing an artificial peptide (10) having a length in the range of 10 to 20 amino acids with an amino acid sequence derived from a portion of a substrate for a protein lysine methyltransferase (PKMT) (2), wherein the peptide (10) has a strong binding affinity to a PKMT (2), comprising the steps of: - Identifying a PKMT peptide substrate with maximum methylatability in a first design process S1 , wherein - in a first sub-step S1.1, a peptide array comprising first peptides having a length of 10 to 20 amino acids with amino acid sequences derived from a section of the substrate for a PKMT is provided, wherein the first peptides have different amino acid sequence variants and each have at least one basic amino acid to be methylated, and methylating the peptides by a PKMT, - in a second sub-step S1.2, at least one first peptide is identified which has a particularly strong methylatability compared to known substrate peptides of a specific protein lysine methyltransferase, - in a third sub-step S1.3, design and production of derived peptides whose amino acid sequence should enable the formation of a loop structure and a basic amino acid to be methylated should be positioned in the center of the loop structure, - in a fourth sub-step S1 .4 testing the methylability of the derived peptides, - Optimizing the binding of the artificial peptides to the PKMT in a second design process S2, where - in a first sub-step 2.1, producing derived peptides from S1, the amino acid sequence of which should enable the formation of a loop structure, wherein a basic amino acid to be methylated should be positioned in the center of the loop structure, and identifying of at least one peptide which has a particularly strong binding compared to known substrate peptides of a particular protein lysine methyltransferase, - in a second sub-step 2.2, design and production of derived peptides based on the data obtained in the previous design process, the amino acid sequence of which should enable the formation of a loop structure, with a basic amino acid to be methylated being positioned in the center of the loop structure, - in a third sub-step S2.3, testing the binding activity of the derived peptides, whereby steps S2.2 and S2.3 are repeated cyclically if necessary, - Optimizing the inhibition of PKMT by the artificial peptides in a third design process S3, where - on the basis of the data obtained in the previous design process, derived peptides are produced whose amino acid sequence should enable the formation of a loop structure, wherein a basic amino acid to be methylated should be positioned in the center of the loop structure, and then the inhibition of the PKMT by the peptides is investigated in order to identify at least one peptide which has a particularly strong inhibition of the PKMT, wherein the third design process can be repeated if necessary. Method according to claim 1, in which a histone protein (1) is selected as the substrate for a PKMT (2). Method according to claim 1 or 2, wherein the basic amino acid is lysine. Method according to one of the preceding claims, wherein the strong binding affinity of the peptide (10) to a PKMT (2) exceeds the binding affinity of a natural histone protein. Method according to one of the preceding claims, wherein a PKMT (2) is selected which, with respect to its enzyme activity, is correlated with a disease. Method according to one of the preceding claims, wherein a PKMT (2) is selected which, with respect to its enzyme activity, is correlated with a cancerous disease. Method according to one of the preceding claims, wherein a PKMT (2) is selected from a group comprising NSD2, SETD2, EZH2, and DOT1 L.An artificial peptide (10) produced by a method according to any one of claims 1 to 7, having a length in the range of 10 to 20 amino acids with an amino acid sequence derived from a portion of a substrate for a protein lysine methyltransferase (PKMT) (2), wherein the peptide (10) has a strong binding affinity to a PKMT (2), characterized in that the amino acid sequence enables the formation of a loop structure, a basic amino acid to be methylated is positioned in the center of the loop structure, and the peptide (10) has an inhibitory effect on the enzyme activity of the protein lysine methyltransferase (2). Peptide (10) according to claim 8, wherein the substrate for a PKMT (2) is a histone protein (1). Peptide (10) according to claim 8 or 9, wherein the basic amino acid is a proteinogenic, non-proteinogenic, or modified proteinogenic amino acid. Peptide (10) according to any one of claims 8 to 10, wherein the basic amino acid is lysine. Peptide (10) according to any one of claims 8 to 11, wherein the peptide (10) is specific for binding by a specific protein lysine methyltransferase (2). Peptide (10) according to any one of claims 8 to 12, wherein the strong binding affinity of the peptide (10) to a PKMT (2) exceeds the binding affinity of a natural histone protein. Use of a peptide (10) according to any one of claims 8 to 13 for inhibiting the enzyme activity of a specific protein lysine methyltransferase (2). Use of a peptide for inhibiting the enzyme activity of a specific protein lysine methyltransferase (2), wherein a peptide obtained in step S1, S2 or S3 of the method according to any one of claims 1 to 7 is used.Peptide (10) according to any one of claims 8 to 13 for use as a medicament. Peptide (10) according to any one of claims 8 to 13 for use in the treatment of cancer, in particular of a cancerous disease in which defective methylation of histone proteins (1) by a protein lysine methyltransferase (2) occurs, wherein the cancerous disease is in particular a cancer of the liver, a cancer of the pancreas, a cancer of the prostate, breast cancer, another solid tumor, or a cancerous disease of the hematopoietic system.

Images