WO2023047376A2

WO2023047376A2 - Peptide constructs for targeted protein degradation

Info

Publication number: WO2023047376A2
Application number: PCT/IB2022/059128
Authority: WO
Inventors: Maria GORNA; Matylda IZERT; Maria KLIMECKA; Patrycja SZYBOWSKA; Anna ANTOSIEWICZ; Piotr TWARDOWSKI
Original assignee: Uniwersytet Warszawski
Priority date: 2021-09-24
Filing date: 2022-09-26
Publication date: 2023-03-30
Also published as: WO2023047376A3; PL439032A1

Abstract

The subject of the invention is a peptide construct comprising a first peptide linked to a second peptide, wherein the first peptide binds a first protein which is degradation target, and the second peptide binds a second protein that is able to initiate degradation of the first protein, wherein the first peptide has a sequence selected from SEQ ID NO. 1-7, and the second peptide has a sequence selected from SEQ ID NO. 8-12 and SEQ ID NO. 28-29. The invention relates also to the use of a peptide having a sequence selected from SEQ ID NO. 1-7 as an anchor in a degrader for targeted protein degradation and the use of a peptide having a sequence selected from SEQ ID NO. 8-12 or SEQ ID NO. 28-29 as a warhead in a degrader for targeted protein degradation.

Description

PEPTIDE CONSTRUCTS FOR TARGETED PROTEIN DEGRADATION

The subject of the invention are molecules for targeted protein degradation (TPD) in bacteria. These molecules belong to a group of molecular tools called degraders or PROTAC® (Proteolysis Targeting Chimera). They are bifunctional, chimeric molecules one end of which binds a target protein, and the other - a component of the proteolytic system present in the bacterial cell, thereby bringing the target protein as a substrate to the protease, causing target protein removal. These molecules can be used to study protein function in the chemical genetics approach, to create artificial systems in the synthetic biology approach, or as a new type of drugs, antibiotics for example.

Drug-induced target protein degradation is a new concept in the drug discovery. Traditionally, drugs modulate the activity of their targets while remain obligatorily bound to the target protein, whereas degradation-inducing ligands act irreversibly by one-time initiation of the degradation process. Thus, one advantage of target protein degradation over the classical targeted mechanism is that a lower drug concentration may be sufficient to induce the desired cellular effects. Targeted degradation was discovered serendipitously, noting that some drugs "accidentally" promote the degradation of their targets by the cellular proteolytic system. Until now, the use of targeted degradation has been limited to human cells. Recently, the antimicrobial anti-tuberculosis antibiotic drug pyrazinamide was found to act as a promoter of its bacterial target protein degradation. The growing antimicrobial resistance makes development of new antibiotics increasingly important (Gopal et al., Progress in Biophysics and Molecular Biology 152 (2020) 10-14).

Taking control of the cellular protein degradation system offers unique drug discovery opportunities, one example of which are proteolysis targeting chimeras (PROTACs). Despite their superior properties in comparison to the classical inhibitors, until now it has not been possible to reprogram the bacterial degradation machinery to interfere with bacterial infections. Small-molecule degraders, so-called BacPROTACs, have been developed that bind to a subunit having ATPase activity (CIpC or ClpC1) that supports the CIpP protease action, preparing neosubstrates for degradation. Degradation assays performed on mycobacteria demonstrate the BacPROTAC in vivo activity, highlighting the potential of this technology to deliver next-generation antibiotics (Morreale et al., https://doi.Org/10,1101 Z2021.06.09.447781 ).

There is still a need in the art for new drugs, such as new antibiotics or other drugs that employ a new mechanism of action.

Thus, the subject of the present invention is a peptide construct comprising: a first peptide linked to a second peptide, whereby the first peptide binds a first protein which is the degradation target, and the second peptide binds a second protein that is able to initiate the degradation of the first protein, wherein the first peptide has a sequence selected from SEQ ID NO. 1-7, and the second peptide has a sequence selected from SEQ ID NO. 8-12 and SEQ ID NO. 28-29.

Preferably, the first peptide and the second peptide are covalently linked to each other.

Preferably, the first peptide and the second peptide are linked to each other by a linker.

Preferably, the linker has a sequence selected from SEQ ID NO. 13-27 or is a non-peptide chain such as a PEG oligomer.

The subject of the invention is also the above-described peptide construct for use as a drug, especially for the treatment of bacterial infections or cancer.

The subject of the invention is also the use of a peptide having a sequence selected from SEQ ID NO. 1-7 as an anchor in degraders for targeted protein degradation.

The invention also relates to the use of a peptide having a sequence selected from SEQ ID NO. 8-12 or SEQ ID NO. 28-29 as a warhead in degraders for targeted protein degradation.

The effects of the degraders according to the present invention are shown in the drawing, where. Fig. 1 shows the effect of degraders with MDFDDDIPF peptide as the warhead on the viability of Escherichia coli bacteria;

Fig. 2 shows the effect of degraders with pyrrhocoricin as the warhead on the viability of Escherichia coli bacteria;

Fig. 3 shows the effect of degraders with NRLLLTG peptide as the warhead on the viability of Escherichia coli bacteria;

Fig. 4 shows the effect of degraders with SWMTTPWGFHLP peptide as the warhead on the viability of Escherichia coli bacteria;

Fig. 5 shows the effect of degraders with SWMTTPWGFHLP peptide as the warhead on the viability of Escherichia coli bacteria in bacterial strains with gene deletion of the proteolytic complex protein components: CIpP protease (Keio ID JW0427), CIpX ATPase (Keio ID JW0428), and SspB adaptor (Keio ID JW0866);

Fig. 6 shows the effect of the degraders with SWMTTPWGFHLP peptide as the warhead on the viability of Escherichia coli bacteria vs. temperature;

Fig. 7 shows the effect of degraders with SWMTTPWGFHLP peptide as the warhead on GroEL protein level.

Fig. 8 shows the effect of degraders with SWMTTPWGFHLP peptide as the warhead on Escherichia coli protein level.

Fig. 9 shows the effect of degraders with cAbGFP peptide as the warhead on the level of RNase E protein in fusion with GFP.

The subject of the present invention are specific "anchor" peptide sequences that can be used to bind the bacterial proteolytic system (for example, to bind CIpXP protease components) in combination in degraders. Also described here are several sequences of example ligands (warheads) that bind target proteins which are of critical importance for the bacteria, wherein the above-mentioned sequences were tested in exemplary degraders with potential antimicrobial activity. In the assays performed, expression of these chimeric degraders decreases the viability of Escherichia coli bacteria, presumably due to efficient degradation of essential proteins needed for normal bacterial cell growth. This makes it possible to introduce the technology of degraders (chimeric peptides) functioning in bacteria for targeted degradation of proteins without the need to modify these proteins.

Authors of the present invention have developed a modular degrader system analogous to the PR.OTAC type by identifying sequences of peptides (so-called anchors) that bind CIpXP protease components for use in degraders. The effectiveness of this technology in Escherichia coli cells is also shown here with several examples of degraders which use these anchors, in combination with sequences that bind various bacterial target proteins involved in selected key processes.

Degraders act differently from traditional inhibitors in that instead of continuously occupying the active site, they only need to cause an irreversible proteolytic event once. Because of this, they act "catalytically" even in small amounts and have broader applications, as they can also remove the “undruggable” proteins that are not enzymes. Published studies of hepatitis C virus-targeting degrader have shown that it is more difficult for the virus to become resistant to this type of compound/mechanism of action. Hence, it is postulated that it will also be more difficult for bacteria to become resistant to the degraders and therefore these degraders will represent an attractive new type of antibiotics. In addition, the above-mentioned degraders can serve as a tool in biology and microbiology to study the protein function and to manipulate of the protein presence.

The present invention uses peptides that bind components of the CIpXP proteolytic system: SspB adaptor, CIpX unfoldases or CIpP peptidases (SspB brings the substrate to the CIpXP complex). Since CIpXP is found in most bacterial species, this approach may have a universal application, also in Gram-negative bacteria. Alternative approaches rely on the recruitment of enzymes that introduce post-translational modifications, such as Arg phosphorylation or pupylation, which are found only in Gram-positive bacteria or Mycobacteria.

The use of degraders that are chimeric peptides can cause their co-degradation together with the target protein, so they may have a weakened (less "catalytic") effect. Improved stability can be achieved by using peptidomimetics.

The effect of the degraders was tested by their induced expression from a plasmid introduced into the bacteria. The length and type of linker connecting parts of the degrader (the anchor to the warhead ligand of the target protein) can affect the properties and efficiency of the degrader, which may need to be optimized in each case.

The anchor sequences according to the present invention recruit the CIpXP system, which is specialized in cytoplasmic protein degradation. Proteins in other compartments (e.g., periplasmic space, bacterial wall or membrane) may be more difficult to access for this type of degraders.

In human cells, the concept of degraders and PR.OTAC technology is excellently developed, and clinical trials of more than a dozen compounds of this type have begun previously, mainly as drugs in anticancer therapies. The action of the degraders in eukaryotic organisms is based mainly on the recruitment of the E3 ubiquitin ligase complex to the target protein, which leads to the ubiquitination of the target protein and its degradation by the proteasome. There is a technical problem standing in the way of transferring this technology to be used with bacteria: the bacteria do not possess the E3 complex, so it is not possible to use E3 protein ligands as anchors to construct bacterial degraders. Due to the absence of the ubiquitin-proteasome system in bacteria, it is therefore necessary to find another way to recruit the proteolytic event (e.g., by binding an enzyme that introduces another post-translational modification to mark proteins for degradation, or by directly binding the protease complex). PR.OTAC type bacterial degraders are neither described nor available at present; the possibility of creating them has been proposed, but their functioning has not been shown in any bacteria.

Pyrazinamide, a natural compound, is known to act on the principle of inducing PanD protein degradation, and is therefore a degrader, but of a different type than PR.OTAC. Pyrazinamide acts on the principle of revealing the presence of a hidden degron and consequently inducing instability of the PanD protein, but as a compound it does not interact directly with the proteolytic system. Pyrazinamide is one of the main drugs used to treat tuberculosis, demonstrating that drugs that induce bacterial protein degradation can be effective antibiotics. With the increasing antibiotic resistance of bacterial pathogens, such kind of degraders antibiotics could provide a range of new antibacterial drugs. In addition, among the research methods to be applied for bacteria, there is no easy method to intervene in the level of protein production at the post-transcriptional level (lack of RNAi type technology). There are known methods for targeting proteins for degradation (including by CIpXP), but such intervention in protein stability or abundance level requires the use of fusion constructs of these proteins, e.g. with added domains or degrons, and thus the introduction of a genetic vector for genetic engineering or protein expression. Using fusion proteins limits the utility of the degron-based method to species susceptible to genetic engineering and under artificial culture conditions. The degrader technology, however, would create entirely new possibilities for studying protein functions at their naturally occurring level and without modifying their sequences or in a wider range of conditions and strains. Due to the existence of the conserved CIpXP pathway also in human mitochondria, degraders targeting proteins to be degraded using the CIpXP pathway may find applications in treating other human diseases, such as cancer.

The introduction of the degrader technology for use in bacteria requires the development of a modular PROTAC type system that could be conveniently designed for the degradation of a selected protein. Such a degrader would consist of:

(i) a well functioning anchor, as the part of the compound that would recruit the protease complex

(ii) which could be connected to a ligand ("warhead") that binds the target protein

(iii) a linker that links the anchor to the (warhead) ligand

In addition, evidence is needed for the action of such constructed degraders leading to the removal of endogenous (naturally expressed, unmodified) bacterial proteins on the principle of inducing a proteolytic event.

Authors of the present invention tested a number of peptide sequences derived from Escherichia coli proteins and confirmed the effective action of the following anchors in the degraders:

• AANDENY peptide (SEQ ID NO. 3) and its doubled length version AANDENYAANDENY (SEQ ID NO. 4) (these are ssrA degron derivatives), binding SspB adaptor

• CYRGGRPALRVVK peptide (SEQ ID NO. 1) and its shorter version ALRWK (SEQ ID NO. 7) and the inverted version KVVRLAPRGGRYC (SEQ ID NO. 2) (these are derivatives of the C-terminal "XB" fragment of the SspB protein), binding CIpX unfoldase) • GIGFGATVK peptide (SEQ ID NO. 5) (IGF loop from CIpX protein) and KSIGLIHQD peptide (SEQ ID NO. 6) (IGF loop from CIpA protein), binding CIpP.

Anchor combinations with different peptide sequences as (warhead) ligands (sequences known from the literature along with the target essential proteins they bind) were tested in the degraders, with the phenotypic effects of bacterial culture growth observed for the following warheads:

• MDFDDDIPF peptide (SEQ ID NO. 8) (C-terminal fragment of SSB protein), which binds replisome proteins involved in DNA replication

• SWMTTPWGFHLP peptide (SEQ ID NO. 9) (Strong Binding Peptide selected by phage display, Chen and Sigler, Cell. 1999;99(7):757-768. doi:10.1016/S0092-8674(00)81673-6), which binds the GroEL protein involved in the protein folding

• NRLLLTG (SEQ ID NO. 10) and VDKGSYLPRPTPPRPIYNRN (SEQ ID NO. 11) peptides and the RRRPRPPYLPRPRPP (SEQ ID NO. 28) derivative, which bind the DnaK protein involved in the protein folding

• DYLDIPAFLR peptide (SEQ ID NO. 12) (C-terminal end of FtsZ protein), which binds divisome proteins involved in bacterial cell division

• cAbGFP (SEQ ID NO. 29) nanobody (Saerens D, Pellis M, Loris R, et al.: Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol. 2005; 352(3): 597-607), directed against GFP, in a strain expressing RNase E fusion with GFP.

Peptide anchors and peptide warheads were linked in different orders (N- or C-terminal anchors) directly after each other or by linker and were tagged with N-terminal myc tag (MEQKLISEEDLGSS). The linker sequence was mostly selected for flexibility, for example a flexible segment [GGS]_nGG (Gly-Gly-Ser repeats, where n = maximally 7) or a rigid segment with alpha helix elements [EAAAK]_m. Degrader-encoding plasmids based on the pBAD vector backbone were introduced into Escherichia coll cells by transformation. The degraders were subjected to induced expression in bacterial cells when arabinose was added to the bacterial medium. Culture density and colony-forming ability were determined for different degraders compared to control constructs (incomplete degraders) and to non-induced cultures to determine the effectiveness of the degraders in reducing bacterial viability. This effect on bacterial growth was interpreted as the result of effective degradation of the target essential proteins. In addition, the levels of target proteins were determined using Western blot and quantitative mass spectrometry techniques.

The anchor sequences according to the present invention can be used in degraders in a fairly universal manner (their effectiveness is shown here for various target proteins and linkers). Specific examples of degraders that are a combination of these anchors with selected peptide warheads are shown, e.g.

• CYRGGRPALRVVK-GGS-SWMTTPWGFHLP

• CYRGGRPALRVVK-(variable length linker)-MDFDDDIPF

• CYRGGRPALRVVK-GGS-

QVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRS SYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS or the use of these peptide warhead examples in degraders that modulate the activity of, for example, the replisome or GroEL and other selected essential proteins and target proteins in fusion with GFP.

Sequences can be used in both peptides and peptidomimetics and include their combinations with, for example, additional factors that increase the efficiency of degrader delivery to bacteria. Linkers can be peptide or other, e.g. PEG chains of selected length. Anchors and warheads can be combined in various ways, e.g., produced/synthesized as a continuous peptide or combined from separately produced parts, e.g., by CLICK chemistry.

EXAMPLES

Example 1

Degrader action study

The effect of the degrader action on bacterial growth was studied using a modified pBAD plasmid having a myc tag-encoding sequence preceding the test peptide-encoding sequence. The plasmids were introduced by transformation into Escherichia coli BW25113 strain. The bacteria were cultured in the presence of ampicillin, which is a selection marker for pBAD plasmids. Overnight bacterial cultures (18 hours at 37°C) were diluted 50,000 times in LB medium supplemented with ampicillin with 0.02% arabinose inducing expression from the pBAD plasmid and as control in medium with no addition of the inducer. Small volumes of diluted cultures were plated onto a 96-well plate and incubated for 16 hours at the appropriate temperature in Tecan Infinite M200 plate reader. The optical density of the culture (absorbance at A = 600 nm) was measured every 30 minutes during the whole measurement period.

In such a way, two series of peptide degraders were tested: the "RepliTAC" degraders using the C-terminal SSB peptide recruiting replisome proteins that bind DNA in the DNA transcription, replication, repair processes (Table 1) and the degraders targeting chaperone proteins (Table 2).

RepliTAC growth curves (Fig. 1)

E. coli BW25113 cells transformed with pBAD plasmids encoding degrader peptides (RepliTACs) using C-terminal SSB peptide against replisome proteins were grown overnight in ampicillin (100 pg/ml) supplemented LB medium at 37°C. The bacteria were then diluted 50,000 times in fresh LB medium with ampicillin (100 pg/ml) and arabinose (0.02%) in a 96-well plate (in triplicates). The plate was then incubated in Tecan M200 plate reader at 30°C with shaking and the OD600 measurements were automatically taken every 30 minutes. The growth curves represent the average value of 3 biological replicates (3 technical repeats each) after background (LB absorbance) subtraction.

The bacteria expressing the XB-linker-SSB construct (RepliTAC) show a decrease in growth in comparison to control (Myc). The effect on OD600 depends on the length of the (GGS)n linker (Fig. 1A). The effect is also visible with invXB as anchor (Fig. 1 B).

Table 1. "RepliTAC" degraders based on C-terminal SSB peptide (SEQ ID NO. 8) targeted against replisome components (DNA-binding proteins involved in DNA transcription, replication and repair), and control peptides

Table 2. Degraders according to the present invention targeted against chaperone proteins, and control peptides

Example 2

Degrader assays against the GroEL and DnaK chaperone proteins

Degraders targeted against chaperone proteins were designed using several different peptides.

The peptides used to construct the degraders against the DnaK protein were the substrate peptide: NRLLLTG (Gragerov A, Zeng L, Zhao X, Burkholder W, Gottesman ME. Specificity of DnaK-peptide binding. J Mol Biol. 1994;235(3):848-854. doi:10.1006/jmbi.1994.1043) and a natural peptide: pyrrhocoricin (Cociancich S, Dupont A, Hegy G, et al. Novel inducible antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus. Biochem J. 1994;300(2):567-575. doi:10.1042/bj3000567). Pyrrhocoricin belongs to a group of natural proline-rich antimicrobial peptides (PrAMPs). These peptides are characterized by good bacterial penetration. Pyrrhocoricin, after penetrating into cells, binds to the DnaK protein reducing its ATPase activity (Kragol G, Lovas S, Varadi G, Condie BA, Hoffmann R, Otvos L. The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry. 2001 ;40(10):3016-3026. doi:10.1021/bi002656a). SBP (Strong Binding Peptide) was identified as a GroEL protein-binding peptide using a phage display method (Chen L, Sigler PB. The crystal structure of a GroEL/peptide complex: Plasticity as a basis for substrate diversity. Cell. 1999;99(7):757-768. doi:10.1016/S0092-8674(00)81673-6). This peptide is a model peptide that mimics the substrate of the GroEL protein, binding to the protein top domain located on the surface of the complex.

The selected peptides were the degrader warheads. They were linked to the XB anchor with linkers of different lengths: GGS, GGSGG or GGSGGSGG and the effect of their expression was tested at 30°C or 42°C.

In the case of the degraders with pyrrhocoricin as the warhead, the toxicity of the warhead alone was so high that it is not possible to say unambiguously whether the slight increase in toxicity of the fusion with the XB anchor could be due to the degradation of the DnaK protein (Fig- 2).

Similarly, the NRLLLTG peptide (hereafter referred to as the NRL peptide) exerted a toxic effect on its own, while its fusion with the XB anchor in degraders produced the opposite effect to the desired one by increasing the bacterial viability (Fig. 3). The observed effect may be related to the interference with the binding of the extended peptide to the DnaK protein.

In the case of the degraders ("GroTAC") targeted against the GroEL protein, no toxicity of the SBP warhead was observed, while its fusion with the XB anchor resulted in a significant decrease in bacterial survival at 42°C (Fig. 4). Comparison of the culture optical density (absorbance at A = 600 nm) (Fig. 4B) with a cell survival assay (BacGIo, a commercial kit from Promega) indicates a bactericidal rather than merely bacteriostatic effect - most cells expressing XB-GGSGGS-SBP degrader died compared to control cultures (Fig. 4C).

Due to the best efficiency of XB anchor with SBP warhead combination, this series of peptides (GroTAC) was selected for further testing of effectiveness of GroTAC degraders in bacterial strains with gene deletion in components of the CIpXP proteolytic complex pathway: CIpP protease (Keio ID JW0427), CIpX ATPase (Keio ID JW0428) and SspB adaptor (Keio ID JW0866). Comparison of the relative final optical density of bacteria at 30°C indicates that the effect observed is CIpP protease-dependent (Fig. 5), confirming the specific and targeted action of GroTAC degraders.

Due to the importance of the chaperone proteins under heat stress conditions, bacterial growth assays were conducted on LB-agar solid medium with ampicillin (drop assay) under different temperature conditions ranging from 18 to 42°C. Increased culture temperature caused a stronger inhibition of the growth of bacteria expressing the GroTAC degrader (whose expression was induced by the presence of 0.02% arabinose in the medium) compared to the control (myc) and non-induced cultures (Fig. 6). This correlation of GroTAC degrader effect with culture temperature indicates that it acts by reducing the level of the GroEL chaperone protein, which is necessary especially with increasing temperature.

In addition, Western blot experiments were performed on protein extracts from bacteria expressing GroTAC degraders or control peptides. Bacteria of wild-type strain BW25113 transformed with the respective pBAD plasmids were cultured in LB medium with ampicillin. When the cultures reached OD600 = 0.1 , arabinose was added to the cultures to a final concentration of 0.02%. Cultures were transferred to 42°C and cultured for additional 4h until the growth curves diverged. The bacteria were centrifuged, and cell pellets were frozen at - 20°C. After thawing, the cell pellets were resuspended in buffer (50 mM Tris pH 7.5, 150 mM NaCI) and sonicated. The soluble protein fraction was recovered by centrifugation of the sonicated bacteria. The obtained extracts were loaded on 12% SDS-PAGE gel at 10 pg per well and electrophoretic separation was performed under denaturing conditions. Western blot analysis was performed. The separated proteins were transferred onto a PVDF membrane (pore size 0.4 pm) by semi-dry transfer in Tris-Glycine buffer with addition of 20% methanol. The membrane was blocked with 3% BSA solution in TBST for one hour at room temperature. It was then incubated overnight with mouse anti-GroEL antibodies in 3% BSA in TBST at 4°C. Excess antibodies were washed off with TBST buffer and then the membrane was incubated with fluorescently labeled anti-mouse secondary antibody Alexa Fluor 488 for one hour at room temperature. After washing off the antibody, the membrane was scanned with a ChemiDoc apparatus (Bio-Rad). The antibody incubation procedure was then repeated with the rabbit anti-enolase antibody and then with the fluorescently labeled anti-rabbit secondary antibody Alexa Fluor 568 (Fig. 7A). Quantitative analysis of the GroEL protein amount in relation to enolase using Image Lab software (Bio-Rad) (Fig. 7B) showed an approximate 40% reduction in GroEL protein level in bacteria expressing XB-GGS-SBP and XB-GGSGGSGG-SBP degraders (GroTACs) compared to bacteria expressing the control peptide (myc).

GroTAC degrader mass spectrometry (Fig. 8)

E. coli BW25113 cells transformed with pBAD plasmids encoding degrader peptides (GroTACs) or control peptides were grown overnight in ampicillin (100 pg/ml) supplemented LB medium at 37°C. The bacteria were then diluted 100 times in fresh LB medium with ampicillin (100 pg/ml) in triplicate and grown to the early exponential phase (OD600 0.1 -0.2). The degrader expression was then induced with 0.02% arabinose and the culture temperature was changed to 42°C. The bacteria were further cultured for further 6 hours and then collected by centrifugation. The bacterial pellets were washed in cold PBS and then kept at -80°C until further processing. Then the bacteria were thawed and lysed. The proteins were fragmented by trypsin digestion and the resulting peptides were labeled with TMTpro™ 16plex Label Reagent Set (Thermo Scientific) according to the manufacturer’s instructions. The labeled peptides were then measured with Dionex UltiMate 3000 nano-LC system coupled to a Q-Exactive HF- X via an EASY-Spray ion source (all Thermo Fisher Scientific). The relative peptide levels were then calculated and compared between the experimental groups. The bacteria expressing GroTAC degrader peptides showed a significant alteration in levels of hundreds of proteins including the GroEL and multiple thermal stress proteins indicating the degradation of the target protein (GroEL). The alteration in levels of other proteins can be assigned to deregulation of bacterial cells resulting from lowered level of the essential GroEL chaperone which is known to regulate stress responses. In the case of control culture samples (XB-GGS or GGS-SBP) their GroEL protein level change was not statistically significant.

Example 3

Degrader against GFP-fusion proteins (Fig. 9)

The degraders against GFP-fusion proteins were created by cloning the codon-optimised gene encoding GFP-nanobody (cAbGFP) (Saerens D, Pellis M, Loris R, et al. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol. 2005; 352(3): 597-607) into pBAD plasmid with myc peptide and the XB anchor at the N-terminus. E coll NCM3416 with endogenous RNase E tagged with GFP on C- terminus (Rne-GFP) were transformed with plasmids encoding Myc-cAbGFP and Myc-cAbGFP- GGS-XB. The transformed bacteria were then cultured overnight in LB medium supplemented with ampicillin (100 pg/ml) at 37°C with shaking. The following day the cultures were refreshed by diluting 100 times in LB medium with ampicillin and grown at 37°C to the mid-exponential phase (OD600 = 0.5-0.6). The bacteria were then induced with 0.1% arabinose and cultured at 30°C. The bacterial samples were collected at 0 min, 15 min, 30 min, 60 min, 120 min and 180 min post induction. The bacteria were collected by centrifugation and the pellets were washed in PBS and stored at -20°C until further analysis. The bacteria were then resuspended in 50 mM Tris pH 8.0 and 150 mM NaCI and lysed by water bath sonication using Bioruptor (Diagenode) on “high” setting with 15 cycles of 30 s on/30 s off intervals. The lysates were cleared by centrifugation and the protein concentration was assessed by Bradford method. The lysates were used to prepare samples for Western blot analysis. The samples containing 15 pg of total protein were denatured by mixing with Laemmli buffer and incubating at 95°C for 5 min. Then, they were loaded on 10% SDS-PAGE gels containing 0.5% 2,2,2-chloroethanol and the electrophoresis was run until the loading dye reached the bottom of the gels. The gels were then activated on ChemiDoc imager (Bio-Rad) at the “stain-free gel” settings. The proteins were then transferred from the gels on the pre-activated PVDF membranes (pore size 0.4 pm) in 1x Tris-Glycine buffer with 10% methanol and 0.05% SDS. The transfer was run at 4°C for 2h at the constant current of 350 mA. The membranes were scanned on ChemiDoc at the “stain-free blot” settings and then blocked in 5% BSA in TBST at room temperature for 2h. The membranes were then cut and incubated with either anti-Myc (Sigma Aldrich) or anti-GFP (Roche) antibodies. The incubation was performed overnight at 4°C with agitation. The membranes were then washed 3 times for 10 min in TBST and incubated for 1 h at room temperature with anti-mouse antibody tagged with Alexa Fluor Plus 647 (Thermofisher Scientific). After washing 3 times for 10 min in TBST the membranes were scanned on ChemiDoc imager. The band intensity and the total protein amount were calculated using ChemiDoc software. The amount of Rne-GFP were normalized by dividing the intensity by the total protein amount. A rapid decrease of the target protein (Rne-GFP) level was observed after the onset of degrader (XB-cAbGFP) induction in comparison to bacteria expressing control peptide (cAbGFP). The reduced levels of Rne-GFP were observed already within 15 minutes and up to 60 minutes after induction. Then the protein level recovered to the starting level.

SEQUENCE LIST

CYRGGRPALRVVK

KVVRLAPRGGRYC

AANDENY

AANDENYAANDENY

<SequenceData sequencelDNumber="5"> GIGFGATVK

KSIGLHQD

ALRVVK

MDFDDDIPF

SWMTTPWGFHLP

NRLLLTG

VDKGSYLPRPTPPRPIYNRN

DYLDIPAFLR

GGGG

GGGGS

GSGS

EAAAK

GGS

GGSGG

<SequenceData sequencelDNumber="19"> GGSGGSGG

GGSGGSGGSGG

GGSGGSGGSGGSGG

GGSGGSGGSGGSGGSGG

GGSGGSGGSGGSGGSGGSGG

GGSGGSGGSGGSGGSGGSGGSGG

EAAAKEAAAK

GGSGGSGGSEAAAKSGGSGGSGG

GGSGGSGGSEAAAKSGGSGGSGGSGG

RRRPRPPYLPRPRPP

Q.VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDAR

NTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQGTQVTVSS

CYRGGRPALRVVKGGSQ.VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAGMSSAGDRSS

YEDSVKGRFTISRDDARNTVYLQM NSLKPEDTAVYYCNVNVGFEYWGQGTQ.VTVSS

</ST26SequenceListing>

Claims

1. A peptide construct containing: a first peptide linked to a second peptide, whereby the first peptide binds a first protein which is degradation target, and the second peptide binds a second protein that is able to initiate the degradation of the first protein, wherein the first peptide has a sequence selected from SEQ ID NO. 1-7, and the second peptide has a sequence selected from SEQ ID NO. 8-12 and SEQ ID NO. 28-29.

2. A peptide construct according to claim 1, wherein the first peptide and the second peptide are covalently linked to each other.

3. The peptide construct according to claim 1, wherein the first peptide and the second peptide are linked together by a linker.

4. The peptide construct according to claim 3, wherein the linker has a sequence selected from SEQ ID NO. 13-27 or is a non-peptide chain such as a PEG oligomer.

5. The peptide construct according to any of the claims 1-4 for use as a drug.

6. The peptide construct according to any of the claims 1-5 for use as a drug to treat either bacterial infections or cancer.

7. Use of a peptide having a sequence selected from SEQ ID NO. 1-7 as an anchor in a degrader for targeted protein degradation.

8. Use of a peptide having a sequence selected from SEQ ID NO. 8-12 or SEQ ID NO. 28-29 as a warhead in degraders for targeted protein degradation.