Peptides and peptidomimetics to destabilize the synaptic complex of a recombinase

Abstract

The invention relates to a peptide or peptidomimetic that reduces and / or inhibits activity and / or efficiency of a recombinase. The invention relates to a peptide comprising an amino acid sequence according to X1SPLX2X3L, wherein X1, X2, and X3 are any amino acid, or a peptidomimetic thereof that binds a target site of a recombinase, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and a length of 7 to 15 amino acids. The invention further relates to an in vitro method for modulating recombinase activity and / or efficiency using the peptide or peptidomimetic according to the invention and to a mixture or composition comprising a recombinase and the peptide or peptidomimetic according to the invention. The invention relates further to a peptide or peptidomimetic according to the invention for use in the treatment of patients in need of antimicrobial therapy and to a pharmaceutical composition comprising the peptide or peptidomimetic according to the invention, preferably for combined administration in combination with an antibiotic.

Description

[0001] PEPTIDES AND PEPTIDOMIMETICS TO DESTABILIZE THE SYNAPTIC COMPLEX OF A RECOMBINASE

[0002] DESCRIPTION

[0003] The invention relates to the field of molecular biology and medicine, more specifically to the field of peptides and peptidomimetics useful in regulating antibiotic resistance and the treatment of patients in need of antimicrobial therapy.

[0004] The invention relates to a peptide or peptidomimetic thereof that reduces and / or inhibits the recombination activity (and / or recombination process or efficiency) of a recombinase. The invention therefore relates to a peptide comprising an amino acid sequence according to X1SPLX2X3L, wherein Xi, X2, and Xs are any amino acid, or a peptidomimetic thereof that binds a target site of a recombinase, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and a length of 7 to 30 amino acids.

[0005] The invention further relates to an in vitro method for modulating the recombination efficiency and / or activity using the peptide or peptidomimetic according to the invention, and to a mixture or composition comprising a recombinase and the peptide or peptidomimetic according to the invention.

[0006] Moreover, the invention relates to a peptide or peptidomimetic according to the invention for use in the treatment of patients in need of antimicrobial therapy, and to a pharmaceutical composition comprising the peptide or peptidomimetic according to the invention with a pharmaceutically acceptable carrier, preferably for combined administration in combination with an antibiotic.

[0007] BACKGROUND OF THE INVENTION

[0008] According to the World Health Organization, an antibiotic resistance crisis is likely and has been recognized as a significant health threat due to its impact on the total number of death cases all around the globe as well as the alarming prognosis of its further increase. Studies have shown that the evolution of antibiotic resistance is being promoted via horizontal gene transfer, implementing plasmids, transposons, and integrons as mobile genetic elements. These rapidly growing antibiotic multi-resistances are particularly relevant for enterobacteria in clinical environments. One of the three major mechanisms of multi-resistance transmittance is based on bacterial integrons. Integrons are DNA elements that are found on plasmids or chromosomes in bacteria. As plasmids, they harvest up to tens of genes that are stored for rapid adaptation, e.g., during antibiotic stress. Despite many research findings over the past decades on the bacterial integron system, various fundamental mechanisms remain elusive. Bacterial adaptation and the spread of antibiotic resistance particularly affect patients with frequent antibiotic treatments, e.g., chronic diseases, and it is at present impossible to inhibit integron adaptation. Integrons are genetic elements able to capture and rearrange gene cassettes. They were first identified in the 1980s on transposons and plasmids in clinical isolates of antibiotic multi-resistant bacteria. Nowadays, it is confirmed that these integrons belong to the class of mobile integrons (Mis) and are recombination platforms representing the major vectors for antibiotic resistance and bacterial adaptation in Gram-negative bacteria. They were found to be associated with many type IV secretion system (T4SS) plasmids, e.g., the prototypical R388. Notably, mobile integrons are species-independent. Thus, the involved recombinase and different genetic elements were found and mixed between species. Besides mobile integrons, chromosomal integrons were discovered in bacterial genomes, like in Vibrio cholera, which mostly contain tens of host-specific genes, often of unknown functions, and tens of adaptation genes.

[0009] The predominant tool for adaptation due to environmental challenges promoted by recombination in Gram-negative bacteria. Mobile integrons are currently structured into five classes; being Class 1 integrons the most common and clinically relevant. Any integron system consists of two major functional elements: 1) a fixed functional platform, with an integrase gene inti and its own promoter (Pint) typically under control of the SOS response protein LexA; a cassette promoter (Pc) and the adjacent primary integration sequence attl, and 2) a cassette library, with promoterless genes packed one after another, flanked by DNA sequences with imperfect inverted repeat sequences called attC sites. During SOS response, the promoters Pint and Pcare accessible (LexA detaches). Thus, inti and the cassettes close to Pccan be transcribed. Genes further away from Pcare less transcribed due to the limited run-length of RNA polymerase.

[0010] However, most cassettes are silent storage units until they participate in a recombination process driven by the recombinase Inti. Inti recognizes two flanking attC sites to excise cassettes from the array and re-integrate them at the primary integration site attl, allowing its subsequent expression. Unlike other DNA recombination systems, Inti recombines single-stranded DNA elements and forms prior to excision (DNA cleavage), a unique complex of four recombinases and two single-stranded DNA (ssDNA) hairpin structures. Before insertion, a complex between one ssDNA hairpin attC site and the double-stranded DNA (dsDNA) insertion site attl is formed. These complexes are called the synaptic complex (excision synaptic complex or insertion synaptic complex, respectively). After synaptic complex formation followed by excision or insertion, the plasmid must first undergo replication to resolve the resulting atypical Holliday junction. After replication, the newly inserted cassettes are accessible for transcription and potentially provide an adaptation solution for the bacteria. Many antibiotic-resistance genes were discovered within integrons, suggesting that the integron is a dominant tool for rapid adaptation in bacteria.

[0011] In the prior art, several methods exist to overcome antibiotic resistance.

[0012] KR101584933 discloses a recombinant vector that inhibits antibiotic resistance by expressing CRISPR-derived RNA (crRNA) and a CRISPR / Cas 9-related protein complex targeting extended- spectrum p-lactamase (ESBL) using a bacterial defense system.

[0013] US9801901 discloses a method to overcome resistance to antibiotics in multidrug-resistant bacteria by contacting the bacteria with an alginate oligomer along with the antibiotic. Additionally, the invention provides alginate oligomers for use in conjunction with antibiotics to treat subjects infected, suspected of being infected, or at risk of infection with multidrug-resistant bacteria, as well as for disinfection and cleaning purposes to combat contamination of sites by such bacteria.

[0014] Marquez et al. (Journal of Clinical Microbiology, vol. 46, no. 10, 3417-3425, 2008) disclose that multiple genetic elements, including class 1 integrons and transposons, facilitate the spread and rearrangement of antibiotic resistance through genetic recombination among bacterial strains causing urinary tract infections in a Uruguayan community.

[0015] W02003087831 A2 discloses a method for diagnosing or monitoring breast cancer by detecting specific BCMP polypeptides or nucleic acids in a patient's biological sample.

[0016] US9801901 B2 discloses an alginate oligomer in combination with an antibiotic to overcome resistance in multidrug-resistant bacteria, applicable on both animate and inanimate surfaces. It discloses a therapeutic use for treating infections in subjects as well as non-medical uses for disinfecting sites contaminated with multidrug-resistant bacteria.

[0017] Thus, multidrug resistance is being addressed through alternative methods like antibacterial peptides and phage therapy. However, these approaches have drawbacks, as antibacterial peptides often cause significant side effects, while phage therapy is highly specific, requiring special phages tailored to specific bacterial strains. Furthermore, there are currently no known inhibitors for the bacterial integron system.

[0018] Hence, an unmet need exists for developing strategies to overcome antibiotic resistance. More specifically, given the current state of the art, there remains a significant demand for improved approaches and techniques in the field to prevent the adaptation and dissemination of antibiotic resistance without disrupting classical bacterial processes.

[0019] SUMMARY OF THE INVENTION

[0020] In light of the prior art, the technical problem underlying the invention was providing alternative or improved means for overcoming antibiotic resistance. The present invention seeks to provide such means while avoiding the disadvantages known in the prior art.

[0021] A further object of the invention was to provide alternative or improved means for the treatment of patients in need of antimicrobial therapy.

[0022] A further object of the invention was to provide means to destabilize a synaptic complex of a recombinase. A further object of the invention was to provide means to reduce and / or inhibit activity and / or efficiency of a recombinase.

[0023] These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

[0024] The present invention, in one aspect, relates to a peptide comprising an amino acid sequence according to

[0025] X1SPLX2X3L (SEQ ID NO. 1), wherein Xi, X2, and Xsare any amino acid, or a peptidomimetic thereof, that binds a target site of a recombinase, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and a length of 7 to 30 amino acids.

[0026] In one aspect, the present invention relates to a peptide comprising an amino acid sequence according to

[0027] X1SPLX2X3L (SEQ ID NO. 1), wherein Xi, X2, and X3 are any amino acid, or a peptidomimetic thereof, that binds a target site of a recombinase, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and has a length of 7 to 15 amino acids.

[0028] In other aspects and embodiments, the peptide or peptidomimetic comprises a sequence according to X1SPX11X2X3L (SEQ ID NO 26), X1SPLX2X3X12 (SEQ ID NO 27), orXiSPXiiX2X3Xi2 (SEQ ID NO. 28), wherein Xi, X2, X3 ,Xn, and X12 are any amino acid.

[0029] Each of the embodiments described herein, in so far as technically compatible with these sequences (SEQ ID NO 1 , 3, 26-28), is considered disclosed in the context of, and may be combined with these sequences.

[0030] In embodiments, any amino acids, variable positions and other features relating to the sequences, or other features of the invention in its entirety, are considered to also relate to embodiments and aspects of SEQ ID NO 26-28, in addition to SEQ ID NO 1 and 3.

[0031] In other aspects and embodiments, the present invention relates to an isolated peptide comprising or consisting of a sequence as described herein, or an isolated peptidomimetic thereof comprising or consisting of a structure corresponding to a sequence as described herein. In other aspects and embodiments, the invention relates to a composition comprising the isolated peptide or isolated peptidomimetic described herein.

[0032] In embodiments, the peptide or peptidomimetic comprises an alpha helix.

[0033] In embodiments, the peptide or peptidomimetic comprises an alpha helix-like structure. In embodiments, the peptide or peptidomimetic comprises and / or resembles the structure of the aN helix of the Inti integrase.

[0034] In embodiments, the peptide or peptidomimetic comprises a length of 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and / or amino acid units (peptide units), preferably a length of 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids and / or amino acid units (peptide units).

[0035] In a preferred embodiment, the peptide or peptidomimetic has a length of 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids and / or amino acid units (peptide units).

[0036] In embodiments, any one or more of the sequences described herein may be combined with the features regarding the length of the peptide or peptidomimetic. For example, in embodiments, the peptide or peptidomimetic as described herein, comprising or consisting of any sequence described herein, may have a length of 7, 8, 9, 10, 11 , 12, 13, 14, or 15 amino acids and / or amino acid units (peptide units). To the knowledge of the inventors, the present invention relates to the first described peptide or peptidomimetic that enables the binding to a target site of a recombinase that consequently leads to the destabilization of said recombinase synaptic complex.

[0037] Furthermore, the present invention represents a novel mechanism to overcome antibiotic resistance by targeting protein-protein interactions essential for recombination, in contrast to existing antibiotics that primarily target bacterial growth processes.

[0038] Numerous antibiotics function by either exterminating or impeding bacterial growth, leading to significant selection pressure and the evolution of resistant strains. However, targeting proteinprotein interactions could undermine the bacteria's ability to exchange resistance genes, thereby slowing the emergence of resistance. Moreover, recombination, which is a fundamental process for bacteria, shares core machinery across different species. Thus, targeting protein-protein interactions within this machinery could have a broad spectrum of activity against various bacteria.

[0039] In embodiments, the peptide or peptidomimetic targets protein-protein interactions of the recombinase. In embodiments, the peptide or peptidomimetic targets protein-protein interactions of the recombinase involved in and / or responsible for recombination.

[0040] In embodiments, the peptide or peptidomimetic compromises the bacteria’s ability to exchange resistance genes. In another embodiment, the peptide or peptidomimetic slows the emergence of bacterial resistance to antibiotics.

[0041] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to

[0042] X1SPLX2X3L (SEQ ID NO 1), wherein X R, K, Q, N, L; X2: D, E, N, S; and X3: A, V, L, I; preferably X R, S; X2: D, S; and X3: R, A.

[0043] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to XISPLX2X3L (SEQ ID NO 1), wherein X R, K, Q, N, L, S, T; X2: D, E, N, S; and X3: A, V, L, I, R, E, G.

[0044] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to XISPLX2X3L (SEQ ID NO 1), wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G.

[0045] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDAL (SEQ ID NO. 2) or a sequence with at least 70%, preferably 80%, sequence identity thereto.

[0046] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDAL (SEQ ID NO. 2).

[0047] In embodiments, the any of the sequences disclosed herein may be modified, for example the “L” of the “SPL” motif in SEQ ID NO 1 may be replaced with an “A”. Such modified sequences encompass any one or more of the sequences described herein, wherein in said sequence the “L” of the “SPL” motif in SEQ ID NO 1 may be replaced with an “A”. ln embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to X1SPX11X2X3L (SEQ ID NO 26), wherein X R, K, Q, N, L, S, T; X2: D, E, N, S; and X3: A, V, L, I, R, E, G; Xn: L, A, I, V.

[0048] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to X1SPX11X2X3L (SEQ ID NO 26), wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G; Xu: L, A.

[0049] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to X1SPLX2X3X12 (SEQ ID NO. 27), wherein X R, K, Q, N, L, S, T; X2: D, E, N, S; and X3: A, V, L, I,

[0050] R, E, G; X12: L, T.

[0051] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to X1SPLX2X3X12 (SEQ ID NO. 27), wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G; X12: L, T.

[0052] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to X1SPX11X2X3X12 (SEQ ID NO. 28), wherein X R, K, Q, N, L, S, T; X2: D, E, N, S; and X3: A, V, L, I, R, E, G; Xn: L, A, I, V; X12: L, T.

[0053] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to X1SPX11X2X3X12 (SEQ ID NO. 28), wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G; Xu: L, A; X12: L, T.

[0054] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to

[0055] (SEQ ID NO. 3) X1SPLX2X3LX4X5X6X7X8X9X10, wherein X R, K, Q, N; X2: D, E, N; X3: A, V, L, I; X4: P, A; X5: P, A; X6: L, O, V, M, A, F; X7: T, S; X8:

[0056] S, T; X9: E, D, Q; and X : R, K, Q, N; preferably RSPLDALPPLTSER (SEQ ID NO. 4) or a sequence with at least 70%, preferably 80%, more preferably at least 90% identity thereto.

[0057] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to (SEQ ID NO. 3) X1SPLX2X3LX4X5X6X7X8X9X10, wherein X R, K, Q, N, S, T, L; X2: D, E, N, S; X3: A, V, L, I, R, E, G; and wherein any one of X4through X10 may be any amino acid.

[0058] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to (SEQ ID NO. 3) X1SPLX2X3LX4X5X6X7X8X9X , wherein X R, K, Q, N, S, T, L; X2: D, E, N, S; X3: A, V, L, I, R, E, G; X4: P, A, M, R, L; X5: P, A, L, Q; X6: L, O, V, M, H, A, F; X7: T, S, I, L, R; X8: S, T, N; X9: E, D, Q, P, G; and X : R, K, Q, N, G.

[0059] In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to (SEQ ID NO. 3) X1SPLX2X3LX4X5X6X7X8X9X , wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G; and wherein any one of X4through Xw may be any amino acid. In embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to (SEQ ID NO. 3) X1SPLX2X3LX4X5X6X7X8X9X10, wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G; X4: P, A, M, R; X5: P, L, Q; X6: L, H, A; X7: S, I, L, R; X8: S, T, N; X9: E, Q, P, G; and X : R, G.

[0060] In one embodiment, the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDALPPLTSER (SEQ ID NO. 4) or a sequence with at least 70%, preferably 80%, more preferably at least 90% identity thereto.

[0061] In one embodiment, the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDALPPLTSER (SEQ ID NO. 4).

[0062] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to TSPADGL (SEQ ID NO. 29).

[0063] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to TSPADGLMLLINQ (SEQ ID NO. 30).

[0064] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to SSPLDAL (SEQ ID NO. 31).

[0065] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to SSPLDALALHLSPG (SEQ ID NO. 32).

[0066] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to LSPLSRL (SEQ ID NO. 33).

[0067] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to LSPLDET (SEQ ID NO. 34).

[0068] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to LSPLDETRQARNG (SEQ ID NO. 35).

[0069] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to SSPLDAL (SEQ ID NO. 36).

[0070] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to LSPLDAL (SEQ ID NO. 37).

[0071] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to RSPLSAL (SEQ ID NO. 38).

[0072] In some embodiments, the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDRL (SEQ ID NO. 39).

[0073] In one embodiment, the peptide or peptidomimetic comprises an amino acid sequence according to RSPADAL (SEQ ID NO. 41).

[0074] In some embodiments, the peptide or a peptidomimetic thereof comprises an amino acid sequence that binds a target site of a recombinase, comprising a sequence as disclosed herein, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and has a length of 7 to 15 amino acids, wherein said peptide does not possess more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 additional amino acids N-terminal of any one of SEQ ID NO 1-41 .

[0075] In some embodiments, the peptide or peptidomimetic comprising SEQ ID NO. 1 , 3, 26-28, does not have more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 additional amino acids at its N-terminal end.

[0076] In embodiments, the peptide or peptidomimetic may comprise or consist of any of the sequences disclosed herein.

[0077] In embodiments, any one of SEQ ID NO 1 , 3, 26-28, may be combined with any other embodiment disclosed herein regarding sequence variation. In embodiments, the variable amino acids of SEQ ID NO 1 , 3, 26-28, at their respective positions, may be combined with (or specified by) any other sequence disclosed herein. For example, the variable amino acids (those defied by X residues) within SEQ ID NO 1 , 3, 26-28, may be defined by one or more of the specific amino acids of any other sequence of SEQ ID NO 1-41 , at their corresponding positions.

[0078] In embodiments, the peptide or the peptidomimetic competes with a native ligand of the recombinase.

[0079] In one embodiment, the peptide or peptidomimetic is designed to mimic a native ligand of the recombinase. In embodiments, the native ligand of the recombinase is the C-terminal domain alpha helix of the recombinase. In another embodiment, the peptide or peptidomimetic mimics the C- terminal alpha helix of the recombinase.

[0080] The C-terminal domain has an unstructured region with a small a-helical structure that takes part in cyclically stabilizing protein-protein interaction (MacDonald et al. 2006; Demarre et al. 2007). It is a specific protein-protein interaction that strengthens the tetrameric assembly in the synapse and is a common feature for tyrosine recombinases (MacDonald et al. 2006; Van Duyne 2007). The C- terminal domains of each integrase subunit interact through a cyclic exchange of small C-terminal a- helices buried in the neighboring subunit. This a-helix is separated from the catalytic core of the C- terminal domain by a long, unstructured flexible chain that is stretched from the subunit reaching the neighboring monomer. The connection is highly ordered, and while the active subunit connects to the inactive one on the same attC site, the inactive subunit reaches across the synapse and connects to the active monomer from the adjacent attC site (Figure 3).

[0081] In embodiments, the peptide or peptidomimetic reduces and / or inhibits the recombination efficiency of the recombinase.

[0082] In another embodiment, the peptide or peptidomimetic decreases the recombination efficiency.

[0083] In embodiments, the peptide or peptidomimetic destabilizes a synaptic complex of the recombinase.

[0084] In embodiments, a synapse assembly is hindered by blocking a C-terminal binding pocket (CtBP) of the recombinase.

[0085] In further embodiments, the peptide or peptidomimetic prevents binding of a C-terminal alpha helix of one recombinase monomer to a binding pocket of a neighboring subunit of another recombinase. In embodiments, the peptide or peptidomimetic prevents the formation of a circular interlocking connection among four monomers of the tetrameric complex of recombinases.

[0086] In embodiments, the peptide or peptidomimetic competes with the native C-terminal helix to bind to the CtBP of the recombinase.

[0087] In embodiments, the peptide or peptidomimetic interacts with a protein according to SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 , SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, and / or SEQ ID NO. 16.

[0088] In embodiments of the invention, the peptide or peptidomimetic interacts with one or more, preferably all residues Y141 , G142, M179, P181 , E182, S183, F283, and / or L287 of the target site of IntH (SEQ ID NO. 8).

[0089] In this context, SEQ ID NO 8 of I ntl 1 is a target, but also a reference sequence for other target sequences, according to e.g., SEQ ID NO 5-7, 9-16. The particular amino acids of SEQ ID NO 8 as shown above, that interact with the agent of the invention, have corresponding amino acids in other sequences of SEQ ID NO 5-7, 9-16, identifiable for example by sequence alignments, and said corresponding amino acids may also interact with the peptide or peptidomimetic of the present invention.

[0090] In embodiments, the peptide or peptidomimetic occupies the CtBP to reduce the mechanical stability of the synaptic complex formation.

[0091] Reducing the recombination efficiency of recombinases offers several promising avenues to combat antibiotic resistance. Firstly, targeting recombinase activity disrupts a pivotal genetic process in bacteria. Recombinases are central in facilitating homologous recombination, a mechanism crucial for exchanging and integrating genetic material, including antibiotic resistance determinants. Directly reducing the recombination function is imperative for impeding the dissemination of resistance. Secondly, inhibition of recombination process effectively neutralizes several mechanisms underpinning horizontal gene transfer, a principal route for acquiring and disseminating antibiotic resistance genes among bacterial populations. By disrupting this process, the ability of bacteria to acquire and disseminate resistance determinants is limited. Moreover, intervention at the level of recombinases may confer a broad-spectrum effect against diverse antibiotic-resistant bacterial strains. Given the fundamental role of recombination in bacterial genetics, targeting this core machinery holds promise for combating resistance across various bacterial species, circumventing the limitations of antibiotics specific to particular bacterial functions. Furthermore, inhibiting recombination may mitigate the development of resistance. In contrast to traditional antibiotics, which impose strong selective pressure on bacterial populations by directly targeting essential cellular processes, recombinase inhibition may not engender the same evolutionary pressure, potentially impeding the emergence of novel resistance mechanisms.

[0092] Thus, in embodiments, the peptide or peptidomimetic prevents the dissemination of antibiotic resistances via horizontal gene transfer of bacterial populations. In other embodiments, the peptide or peptidomimetic confers a broad-spectrum effect against antibiotic-resistant bacterial strains.

[0093] In further embodiments, the peptide or peptidomimetic inhibits the antibiotic resistance of various bacterial species. In embodiments, the peptide or peptidomimetic mitigates the development of resistance. In further embodiments, the peptide or peptidomimetic impedes the emergence of novel resistance mechanisms.

[0094] In embodiments, the recombinase comprises an amino acid sequence according to

[0095] (SEQ ID NO. 5) FATX1LLX2X3G, wherein X H, A; X2: E, R; X3: V, S, preferably (SEQ ID NO. 6) FATHLLEVG or (SEQ ID NO. 7) FATALLRSG, or a sequence with at least 70%, preferably 80%, more preferably at least 90% identity thereto.

[0096] In embodiments, the recombinase comprises an amino acid sequence according to

[0097] Yi3oGi3i ....xi72l— F273ATX2LLX3X4G, wherein X E, S; X2: H, A; X3: E, R; X4: V, S, with reference to SEQ ID NO. 11 and / or corresponding amino acids from SEQ ID NO. 12 to SEQ ID NO. 17.

[0098] In embodiments, the amino acid sequence according to Y130G131— X172i — F273ATX2LLX3X4G, wherein X E, S; X2: H, A; X3: E, R; X4: V, S, with reference to SEQ ID NO. 11 and / or corresponding amino acids from SEQ ID NO. 12 to SEQ ID NO. 17, forms the cavity for proline and / or interacts with serine of the peptide or peptidomimetic of the present invention. In embodiments, the binding pocket of I ntl4 comprises an amino sequence according to

[0099] R108DEIRRLL115- K127LLYGSG131- — T168LAKELY174— - F273ATHLLEVG281, which interacts with the peptide or peptidomimetic of the present invention.

[0100] Table 1. Preferred amino acid and nucleotide sequences of the present invention

[0101] In embodiments, the recombinase is a tyrosine recombinase.

[0102] In embodiments, the peptide or peptidomimetic inhibits site-specific recombination.

[0103] Tyrosine recombinases (YRs) are enzymes crucial for mediating site-specific DNA recombination, playing an essential role in various genetic processes. In the context of antibiotic resistance, YRs are particularly significant due to their involvement in the mechanisms of horizontal gene transfer. Through horizontal gene transfer, bacteria can acquire and disseminate genetic determinants, including antibiotic-resistance genes, thus contributing to the spread of antibiotic resistance within bacterial populations. YRs facilitate the integration of foreign DNA, often harboring antibiotic- resistance genes, into the bacterial chromosome, ensuring a stable inheritance of resistance traits. Moreover, YRs play a pivotal role in the functionality of integrons, specialized DNA structures that capture and rearrange antibiotic-resistance genes. By mediating cassette capture, YRs dynamically enrich the pool of resistance genes within bacteria, allowing for adaptation to various antibiotic pressures. In embodiments, the peptide or peptidomimetic disrupts antibiotic resistance mechanisms. In further embodiments, the peptide or peptidomimetic hinders horizontal gene transfer. In embodiments, the peptide or peptidomimetic hinders the integration of foreign DNA into the bacterial chromosome. In embodiments, the peptide or peptidomimetic disrupts the functionality of integrons.

[0104] In embodiments, the recombinase is an integrase, preferably a bacterial integrase.

[0105] In embodiments, the recombinase is an Inti integrase, a phage lambda integrase, HP1 integrase, Bacteriophage P22 integrase, Bacteriophage PF1 integrase, a cyclic recombinase (Cre), Cre P1 or any directed evolution derivatives, XerC / D recombinase, XerH, XerS and XerA recombinases, or a Flp recombinase.

[0106] In one embodiment, the peptide or peptidomimetic destabilizes an Inti integrase, preferably an Inti 1 and / or and Intl4 integrase.

[0107] The integrase gene inti is a part of the integron functional platform that encodes a protein from the A phage family of tyrosine recombinases - the integrase Inti. Different integron integrases have a high sequence similarity, indicative of 3D structural resemblance. In particular, the catalytic domain is highly conserved, dictating that a tetramer is required to perform the recombination reaction. The bacterial integron integrase has evolved the ability to use non-canonical substrates: Inti preforms recombination by interacting with two different types of target sites: (i) attC x attC for cassette excision, where attC sites are formed by an ssDNA hairpin and (ii) attl x attC for the insertion of a circular gene-carrying fragment, where attl is a prototypical double-stranded DNA recombination site, and attC is again a DNA hairpin site. While attC sites carry distinct secondary structure and sequence elements, attl sites exhibit no particular secondary structure elements, showing the remarkable ability of Inti to recognize both targets. DNA cleavage by the active tyrosine occurs for Inti only in the synaptic complex, formed by four monomers and two att sites. Noteworthy, besides four integrases being involved, only two monomers bound to the R-box are active and will cleave the DNA substrate, while the two other monomers are inactivated. Similar stoichiometry and mechanism are seen in other Tyr-recombinases.

[0108] Thus, the peptide or peptidomimetic described in the invention demonstrates unexpected effectiveness not only in targeting the bacterial integron system but also in addressing various other tyrosine recombinases.

[0109] In embodiments, the peptide or peptidomimetic invention prevents DNA cleavage.

[0110] In embodiments, the peptide or peptidomimetic induces a reduced disassembly force (preferably using a single-molecule optical tweezers stability assay, as shown below) of below 10, 9, 8, 7, 6, 5, 4 pN, preferably below 10, 9, 8,7 pN. The disassembly force may also fall within a range formed between any two values (as endpoints) mentioned in the list. ln another aspect or in embodiments of the invention described herein, the invention relates to an in vitro method for modulating recombinase activity and / or efficiency using the peptide or peptidomimetic according to the invention. In another aspect or in embodiments of the invention described herein, the invention relates to a mixture or composition comprising a recombinase according to the invention and the peptide or peptidomimetic according to the invention.

[0111] In another aspect or in embodiments of the invention described herein, the invention relates to a peptide or peptidomimetic according to the invention for use in the treatment of patients in need of antimicrobial therapy.

[0112] In embodiments, the peptide or peptidomimetic thereof inhibits antimicrobial resistance.

[0113] In embodiments, the peptide or peptidomimetic is intended for use according to the invention, wherein the patient a. has a chronic infection, preferably osteomyelitis, endocarditis, cystic fibrosis, tuberculosis, sinusitis, skin infections, ear infections, Lyme disease, urinary tract infections, and / or prosthetic joint infection; and / or b. has an immunocompromised medical condition; and / or c. will undergo or has undergone a surgery.

[0114] In another aspect or in embodiments of the invention described herein, the invention relates to a pharmaceutical composition comprising the peptide or peptidomimetic according to the invention with a pharmaceutically acceptable carrier.

[0115] In embodiments, the pharmaceutical composition is administered to a patient in combination with an antibiotic. In embodiments, the pharmaceutical composition is administered to a patient in combination with one or more antibiotics.

[0116] Inhibiting protein-protein interactions might be most effective when combined with antibiotics. This dual approach could weaken the bacteria and reduce the chance of adaptation.

[0117] In embodiments, the pharmaceutical composition reduces or reduces the risk of bacterial resistance to one or more antibiotics.

[0118] The various aspects of the invention are unified by, benefit from, are based on and / or are linked by the structural and / or functional features, including functional properties and beneficial technical effects, of the peptide or peptidomimetic described herein. The features disclosed in the context of the peptide also apply to and are considered disclosed in the context of the peptidomimetic, and vice versa. Any features disclosed in any further aspects of the invention, such as the in vitro method, the mixture or composition, the pharmaceutical composition, and the medical use thereof are also considered disclosed in the context of the peptide or peptidomimetic and vice versa.

[0119] DETAILED DESCRIPTION OF THE INVENTION

[0120] The present invention relates to a peptide comprising an amino acid sequence as described herein, or a peptidomimetic thereof that binds a target site of a recombinase, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and has a length of 7 to 30, 7 to 20, 7 to 15, or 7 to 14 amino acids.

[0121] As used herein, a "peptide," "polypeptide," "polypeptide fragment," and "protein" are used interchangeably unless specified to the contrary and according to conventional meaning, i.e. , as a sequence of amino acids. Polypeptides are not limited to a specific length, e.g., they may comprise a full-length protein sequence or a fragment of a full-length protein and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0122] Protein or peptide sequence modifications, which may occur through substitutions, are also included within the scope of the invention. Substitutions, as defined herein, are modifications made to the amino acid sequence of the protein, whereby one or more amino acids are replaced with the same number of (different) amino acids, producing a protein that contains a different amino acid sequence than the primary protein. Substitutions may be carried out that preferably do not significantly alter the function of the peptide. Like additions, substitutions may be natural or artificial. It is well known in the art that amino acid substitutions may be made without significantly altering the protein's function. This is particularly true when the modification relates to a "conservative" amino acid substitution, substituting one amino acid for another of similar properties. Such "conserved" amino acids can be natural or synthetic, which, because of size, charge, polarity, and conformation, can be substituted without significantly affecting the structure and function of the protein. Frequently, many amino acids may be substituted by conservative amino acids without deleteriously affecting the protein's function.

[0123] In general, the non-polar amino acids Gly, Ala, Vai, lie, and Leu; the non-polar aromatic amino acids Phe, Trp, and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gin, Asn, and Met; the positively charged amino acids Lys, Arg and His; the negatively charged amino acids Asp and Glu, represent groups of conservative amino acids. This list is not exhaustive. For example, it is well known that Ala, Gly, Ser, and sometimes Cys can substitute for each other even though they belong to different groups.

[0124] Conservative amino acid substitutions are not limited to naturally occurring amino acids but also include synthetic or non-proteinogenic amino acids.

[0125] Commonly used synthetic amino acids are omega amino acids of various chain lengths and cyclohexyl alanine, which are neutral non-polar analogs; citrulline and methionine sulfoxide, which are neutral non-polar analogs, phenylalanine, which is an aromatic neutral analog; cysteic acid which is a negatively charged analog and ornithine which is a positively charged amino acid analog. Like the naturally occurring amino acids, this list is not exhaustive but merely exemplary of the well- known synthetic amino acids in the art.

[0126] Sequence variants of the claimed peptides and peptidomimetics, for example those defined by % sequence identity, that maintain similar binding properties of the invention, are also included in the scope of the invention. Such variants, which show alternative sequences, but maintain essentially the same properties as the specific sequences provided are known as functional analogs, or as functionally analogous. Sequence identity relates to the percentage of identical nucleotides or amino acids when carrying out a sequence alignment. The recitation "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Included are peptides and peptidomimetics having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences described herein, preferably where the variant maintains at least one biological activity of the specific peptide, such as binding or recombinase destabilization, as described herein. Any suitable methods of alignment of sequences for determination the percent sequence identity may be employed and are known to a person skilled in the art. The determination of percent identity between any two or more sequences can be accomplished using a publicly available mathematical algorithm.

[0127] As used herein, the term “peptidomimetic” refers to a compound whose pharmacophore mimics a peptide or protein in 3D space with the ability to interact with a designated biological target and produce in essence the same or similar biological effect. A pharmacophore is typically defined as a collection of steric and electronic features that is necessary to ensure supramolecular interactions between a compound and a target, and preferably to interact with, trigger, block inhibit, or otherwise modulate, a biological function.

[0128] In embodiments, the peptidomimetic comprises a plurality of amino acid residues joined by a plurality of peptide bonds, with one or more structural changes over naturally occurring amino acid residues and / or peptide linkages. In embodiments, peptidomimetics are protein-like chains designed to mimic peptides, either by modifying existing peptides or by designing similar systems, such as peptoids and p-peptides. These alterations can optimize molecular properties like stability and biological activity, facilitating the development of drug-like compounds.

[0129] Four classes of peptidomimetics are identified based on their similarity to precursor peptides (Classes A-D) (see Peptidomimetic toolbox for drug discovery; Chem. Soc. Rev., 2020,49, 3262- 3277). Class A closely resembles natural peptides, featuring mainly proteogenic amino acids, while Class B involves more significant modifications, including non-natural amino acids. Class C comprises highly modified structures replacing the entire peptide backbone with small molecules. Class D consists of small molecule drugs mimicking a peptide mode-of-action.

[0130] In one embodiment, the peptidomimetic is a Class A peptidomimetic. In another embodiment, the peptidomimetic is a Class B peptidomimetic. In another embodiment, the peptidomimetic is a Class C peptidomimetic. In another embodiment, the peptidomimetic is a Class D peptidomimetic. In embodiments, the peptidomimetic is a Class A, Class B, Class C, or a Class D peptidomimetic.

[0131] Structural and functional studies of the proteins have suggested that protein-protein interactions (PPI) are required for various biological processes. PPI may be stable or transient and reversible. The amino acids that are present on the surface of proteins provide them with high specificity and affinity in their binding character. Since PPI surfaces are made up of epitopes of amino acids, peptides or peptidomimetics are a relevant choice to modulate such interactions. Peptidomimetics act as a mimicking surface of one of the proteins, can interfere with PPI and modulate biological activity of a target.

[0132] In embodiments, peptidomimetics are employed to improve the half-life and / or bioavailability of the compound. For example, to overcome short half-life and low bioavailability, several strategies have been investigated that can be adopted in the design of peptide-based drugs (Peptides and peptidomimetics as immunomodulators; Immunotherapy. 2014; 6(6): 755-774; Peptidomimetic toolbox for drug discovery, Chem. Soc. Rev., 2020,49, 3262-3277). In vivo stability of peptides can be enhanced by peptide backbone modification; this can be accomplished by introduction of unnatural amino acids or D-amino acids, peptide-bond modification, N- and C-termini modifications and / or constraining the backbone by introducing cyclization, resulting in molecules that are stable against enzymatic degradation. Bioavailability and renal clearance problems can be overcome by PEGylation of the peptides.

[0133] Speaking generally, modification of a peptide backbone and / or side chain of amino acid residues can produce a peptidomimetic. Peptidomimetics are typically understood as compounds whose pharmacophore mimics a natural peptide or protein in 3D space with the ability to interact with the biological target and produce the same biological effect (Peptidomimetics, a synthetic tool of drug discovery, Curr Opin Chem Biol. 2008 Jun;12(3):292-6). Proteins exert their biological effects through small regions on their surface called epitopes. A short sequence of peptide or functional groups that are close together can be reproduced in smaller, conformationally similar fragments that can bind to a target site and provide steric inhibition between a target site and the native protein ligand. Peptidomimetics typically have advantages over peptides in terms of stability and bioavailability associated with a natural peptide. In terms of design considerations, peptidomimetics can be designed from protein epitopes with global or local conformational restrictions (Immunotherapy. 2014; 6(6): 755-774).

[0134] Global conformational restrictions typically impose a particular shape or secondary structure on the peptide and also provide stability against enzymatic degradation. In embodiments, examples of global conformational constraints include cyclization of the peptide using nonpeptide moieties, lactam bridges or inclusion of penicillamine (dimethyl cysteine) to form disulfide bonds. In embodiments, local conformational restrictions can be applied using backbone modifications at particular amino acid residues or between two amino acid residues in the peptide. In embodiments, backbone amides can be replaced by amide bond-like surrogates and isosteric substituents. In embodiments, backbone-modified mimetics can have regular amino acids or synthetic or modified amino acids. In embodiments, side chains of amino acids in the peptides can be replaced with analogs of amino acids that have functional properties similar to those of amino acid side chains but with conformational restrictions of x angles for side-chain rotation. In embodiments, the side chain- modified peptidomimetics can expose the proper functional groups to bind with the target site of the recombinase with high affinity, similar or improved compared with normal side chains of amino acids. In embodiments, a secondary structure of the peptide is mimicked using a-helical, p-turn or p-strand constraints. In embodiments, the entire peptide backbone can be modified to mimic turn or helical structures using organic functional groups without any peptide bonds. In embodiments, the peptidomimetic is designed to maintain the helical structure of the peptides described in the examples below. Means for maintaining the helical structure of a peptide are known in the art (Peptidomimetic toolbox for drug discovery, Chem. Soc. Rev., 2020,49, 3262-3277). For example, Mimetics of helices can be obtained by routine procedures. A helix structure is one of the most common peptide secondary structures, constituting more than 30-40% of the polypeptide structure in proteins. They are formed by repetitive sequences stabilized by intramolecular hydrogen-bonds between sequential residues. Usually, they are classified depending on the number of residues participating in one turn and on the number of atoms between the carbonyl group of the amino acid at position i and the amide proton at position i + n. The most widely occurring helix in globular proteins (around 90%) is the 3.613-helix, better known as a-helix, and 10% of other helices are constituted by the 310-helix. Other less common examples of helical conformations are the 14- helix (314-helix) or the 12-helix (2.512-helix). As for the relevance of the a-helix in modulating protein-protein interactions, different approaches have been reported to develop more stable and constrained a-helix mimetics, since the early 1980s. For example, peptoids are known a-peptide mimetics, in which the side chains are attached to the amide nitrogen atom rather than to the a- carbon atom, thus resulting in a formal shift of the side chain position, with a significant improvement of the resistance to enzymatic hydrolysis and cell permeability. While peptoids possess a peptide character, another strategy for designing helix mimetics is the use of molecular scaffolds capable of replacing the essential conformational components of helices.

[0135] As used herein, the term “amino acid unit”, also known as “peptide unit”, relates to a sequence of atoms in a peptide or peptidomimetic chain that may be represented by the general formula -CHR-CO-NH-, where R denotes the side chain of an amino-acid residue, or peptidomimetic structure analogous to said formula. In the context of a peptidomimetic, even if changes to the structure are evident, compared to a naturally occurring peptide, the basic unit of an amino acid, or peptide unit, can be identified without undue effort by a skilled person. As described herein, the length of the peptide may, in embodiments, be defined by amino acids, or amino acid units, whereby the amino acid unit or modified amino acid unit of a peptidomimetic can be determined as a measure of peptide or peptidomimetic length. For example, backbone modifications may be carried out in peptidomimetics, but the length of the peptide may still be identified based on the number of amino acid units. With reference to Immunotherapy. 2014; 6(6): 755-774, non-limiting examples of backbone peptide modifications in mimetics may be one or more of the following example di-peptide mimetic structures, wherein R, R1 and R2 and / are amino acid side chain structures (whereby the first structure represents an unmodified peptide; di-peptide of two amino acid units -CHR-CO-NH-CHR-CO-NH-): “Recombinases” are enzymes involved in genetic recombination, facilitating nucleic acid exchange reactions between sequences. Originating from diverse organisms, including bacteria, phages (such as bacteriophages), fungi, and archaea, these enzymes play critical roles in various molecular biological processes. They facilitate DNA repair mechanisms, regulate gene expression patterns, and enable precise genome manipulation. Notably, tyrosine recombinases represent a subset of recombinases characterized by utilizing an active site tyrosine nucleophile. Widely distributed among prokaryotes and their viruses, with emerging evidence of their presence in various eukaryotic organisms, tyrosine recombinases exhibit remarkable sequence diversity and possess distinct functional domains for specific DNA recognition and binding.

[0136] Recombinases play a crucial role in DNA rearrangements, facilitating various vital biological processes. The archetypal example of the mechanism is the integration of the lambda bacteriophage genome into the Escherichia coli chromosome (Campbell 1962). Beyond the integration and removal of viral genomes from host chromosomes, site-specific recombination is employed for multiple purposes. It takes part in cell division, resolving catenated DNA circles and regulating plasmid copy numbers (Ip et al. 2003), modulates gene expression by flipping DNA segments to either activate or deactivate genes, and it fosters genetic diversity by acquiring and integrating gene cassettes and facilitating conjugative transposition (Van Duyne 2007; Meinke et al. 2016; Landy 2015). In each case, recombinase enzymes recognize specific DNA sequences and facilitate DNA rearrangements at the site. Non-limiting examples of members of this recombinase families are:

[0137] • The phage A integrase is accountable for integrating the A phage genome (Landy 2015; Kwon et al. 1997).

[0138] • The phage P1 Cre recombinase cyclizes the P1 genome and resolves genomic multimers (Meinke et al. 2016; Gibb et al. 2010).

[0139] • The E. coli XerC / XerD recombinases resolve dimers of the bacterial chromosome (Castillo, Benmohamed, and Szatmari 2017; Subramanya et al. 1997).

[0140] • The yeast Flp recombinase regulates plasmid copy numbers (Chen et al., 2000; Jayaram et al., 2015).

[0141] Members of the tyrosine recombinase family possess a distinctive ability to conduct a complete sitespecific recombination process between two DNA molecules without the need for high-energy cofactors (Meinke et al. 2016; Esposito and Scocca 1997; Van Duyne 2007).

[0142] Recombinases, particularly “tyrosine recombinases,” play pivotal roles in molecular biology and genetics, orchestrating site-specific DNA recombination events critical for various biological processes. Tyrosine recombinases, such as lambda Int protein, Cre recombinase, XerC / XerD proteins, and Flp protein, mediate the recombination of DNA duplexes by catalyzing two consecutive strand breakage and rejoining steps. They also facilitate the topoisomerization of reactants, contributing to DNA repair, gene expression regulation, and genome manipulation. These enzymes exhibit diversity in sequence and function, with conserved catalytic residues clustered in the C- terminal domain, including an active site tyrosine nucleophile. Structural studies reveal a common three-dimensional fold among tyrosine recombinases, reflecting their evolutionary conservation despite sequence diversity. The recombination reactions proceed via a covalent intermediate, where the tyrosyl residue esterifies the DNA 3'-phosphoryl group, highlighting their role as the tyrosine recombinase family. Evolutionary analysis suggests integrases aggregate into subfamilies based on biological roles, with prokaryotic and eukaryotic recombinases exhibiting distinct characteristics. Tyrosine recombinases also contribute to bacterial adaptation and antibiotic resistance by facilitating genome rearrangements and horizontal gene transfer through mobile genetic elements.

[0143] In embodiments, the peptide or peptidomimetic of the present invention binds a target site of a recombinase or a homolog thereof. In embodiments, the peptide or peptidomimetic prevents and / or slows down bacterial adaptation. In embodiments, the peptide or peptidomimetic inhibits genome rearrangements. In further embodiments, the peptide or peptidomimetic inhibits horizontal gene transfer, preferably through mobile genetic elements.

[0144] “Integrases” are enzymes that catalyze site-specific recombination between DNA sequences known as attachment (att) sites, independent of external energy and without gaining or losing nucleotides. They belong to two main recombinase superfamilies: tyrosine and serine integrases, distinguished by the amino acid residue involved in catalysis. Tyrosine integrases generally have seven canonical conserved amino acid residues thought to play a catalytic role in recombination reactions: Arg (Rl), Glu / Asp (E / DII), Lys (Kill), His (HIV), Arg (RV), His (HVI), and Tyr (YVII). Tyrosine, the essential characteristic residue, can directly attack the phosphodiester bonds of target DNA (att sites), forming a covalent 3' phosphotyrosine high-energy intermediate. In general, the catalytic region of tyrosine integrases is located in the C-terminal domain, while the binding region is located in the N-terminal domain. The N-terminal domains are frequently variable, while C-terminal domains are relatively conserved among different tyrosine integrase families. The seven typical conserved amino acid residues are all in the C-terminal domains. Integrases play diverse roles in molecular biology, facilitating genome rearrangements, gene regulation, and horizontal gene transfer, and are widely used in genetic engineering applications for gene integration, knockout, and modification. Nonlimiting examples of integrases include Inti integrases, phage lambda integrases, HP1 integrases, Bacteriophage P22 integrases, Bacteriophage PF1 integrases, a cyclic recombinases (Cre), Cre P1 or any directed evolution derivatives, XerC / D recombinases, XerH, XerS and XerA recombinases, or Flp recombinases.

[0145] “attC sites” flank open reading frames (ORF), providing the integrase a universal marker for excision and insertion. However, unlike the attl site, where most insertion events take place, attC regions participate in the recombination reaction as a single-stranded DNA (ssDNA) folded into an imperfect hairpin structure. To date, more than 200 attC sites have been discovered in more than 400 bacterial species. Even though attC sites display high sequence and size diversity, ranging from ~60 to ~150 nucleotides (nts), they are recognized by the integrase due to a common set of characteristics. All attC sites are comprised of a (partial) palindromic sequence being able to form a hairpin-like structure. Once folded as a hairpin, the attC sites consist of two integrase binding sites in opposite orientations, the “L-box” (formed by L’ and L” pairing) and the “R-box” (formed by R’ and R”) that are separated by an unpaired central spacer (UCS), two to three extrahelical bases (EHBs) and a variable terminal structure (VTS) of varying size, structure, and yet unknown function. The R-box is the location of the recombination event, more precisely - the highly conserved 5’-AAC-3’ triplet among all attC sites. Various studies have identified important sequence - and, by consequence - structural elements for the attC sites to show efficient recombination. Among the most important identified sequence elements are the UCS, the EHBs, and a recently discovered high GC content near the apical loop region. As a consequence, attC sites not only provide a recombination substrate for the integrase but also ensure that the subsequent integration will insert the ORF in the correct orientation relative to the promoter Pc.

[0146] In embodiments, the peptide or peptidomimetic reduced and / or inhibits the recombination activity and / or efficiency of the integrase.

[0147] The “synaptic complex” of recombinases refers to a specialized protein-DNA structure formed during genetic recombination, which is crucial for various biological processes. The synaptic complex of recombinases shares common features irrespective of the specific recombinase type. Composed of multiple subunits of the recombinase enzyme bind to two DNA substrates (coming from the same molecule or different molecules). This binding brings the DNA molecules into close alignment, facilitating strand exchange. The formation of the synaptic complex is a crucial step in the recombination process, ensuring the correct DNA molecules are brought together, and the recombination event occurs at the desired location.

[0148] Tyrosine recombinases form a synaptic complex through protein-protein interactions within the recombinase. For instance, Cre recombinase exhibits a tetrameric structure bound to two DNA substrates, facilitating strand exchange.

[0149] In embodiments, the peptide or peptidomimetic prevents the formation of the synaptic complex. In embodiments, the peptide or peptidomimetic inhibits protein-DNA formation during recombination. In embodiments, the peptide or peptidomimetic inhibits strand exchange of DNA molecules.

[0150] The “C-terminal domain” or “C-terminal tail” of a recombinase has an unstructured region with a small a-helical structure that takes part in cyclically stabilizing protein-protein interaction (MacDonald et al. 2006; Demarre et al. 2007). It is a specific protein-protein interaction that strengthens the tetrameric assembly in the synapse and is a common feature for tyrosine recombinases (MacDonald et al. 2006; Van Duyne 2007). The C-terminal domains of each integrase subunit interact through a cyclic exchange of small C-terminal a-helices buried in the neighboring subunit. This a-helix is separated from the catalytic core of the C-terminal domain by a long, unstructured flexible chain that is stretched from the subunit reaching the neighboring monomer. The connection is highly ordered, and while the active subunit connects to the inactive one on the same attC site, the inactive subunit reaches across the synapse and connects to the active monomer from the adjacent attC site (Figure 3). It is not known whether the C-terminal connection exists on the dimer level for integron integrases without the synaptic complex assembly. However, for other members of the tyrosine recombinase family, it was shown that such a connection exists and seems to be a conserved stabilizing feature of the synaptic complex (Hickman et al. 1997). In the case of Cre protein, the dimer bound to the DNA substrate also demonstrated C-terminal helix interaction, but it was not reciprocal, perhaps due to the slightly bent substrate conformation. The connection between N- and C-terminal domains in neighboring subunits indicates cooperative binding for Cre proteins and the XerCD systems (Van Duyne 2007). For the bacteriophage HP1 integrase, the C-terminal helix swap is symmetrical and reciprocal. Apart from the stabilizing role, it is proposed that the C-terminal pocket binding also provides important mechanistic control of the cleavage reactions. The reason behind this lies in the position of attacking tyrosine, which is in the C-terminal domain very close to the flexible linker that carries the small connecting a-helix. It is suggested that alterations in the quaternary structure of the recombination complex may be detected by the catalytic tyrosine via the linker. This creates a stereochemical connection between the overall arrangement of the synaptic complex and the placement of a catalytic amino acid. The overall conservation of the C-terminal cyclic connection and the stabilization effects are important for maintaining recombination fitness in vivo, as was shown for the E. coli XerCD system (Subramanya et al. 1997; Van Duyne 2007).

[0151] Mutations in either the C-terminal helix or the acceptor pocket reduced recombination efficiency (Van Duyne 2007). It is so far uncertain if such stabilizing and allosteric interactions are valid for all members of the tyrosine recombinase family. However, as the C-terminal domain is generally very similar in structure between different family members, it is undoubtedly a common functional stabilization and regulation mechanism (Meinke et al. 2016; Landy 2015; Jayaram et al. 2015). Noteworthy, in the case of the integron integrase, the C-terminal helix interaction happens between the same domains of the monomers as the receiving pocket is located on the C-terminal part as well (MacDonald et al. 2006). From the crystallographic structure, a slight difference can be observed between interactions of cis (active-to-inactive pair) versus in trans (inactive-to-active pair) modes. The location of the binding pocket remains the same, but the molecular interactions upon binding differ.

[0152] In embodiments, the peptide or peptidomimetic of the present invention weakens the tetrameric assembly in the synapse. In embodiments, the peptide or peptidomimetic prevents binding of an active subunit of the recombinase to the inactive one on the same attC site. In embodiments, the peptide or peptidomimetic prevents binding of an inactive subunit of the recombinase to the active monomer from the adjacent attC site. In embodiments, the peptide or peptidomimetic prevents binding of an active subunit of the recombinase to the inactive one on the same attC site and the binding of an inactive subunit of the recombinase to the active monomer from the adjacent attC site.

[0153] In embodiments, the peptide or peptidomimetic inhibits the formation of cis modes. In embodiments, the peptide or peptidomimetic inhibits the formation of trans modes.

[0154] The binding of the peptide or peptidomimetic thereof to a recombinase can be assessed by various “detection methods.” Non-limiting examples of detection techniques include Forster Resonance Energy Transfer (FRET), Fluorescence Correlation Spectroscopy (FCS), Electrophoretic Mobility Shift Assay (EMSA), Co-immunoprecipitation (Co-IP), Microscale Thermophoresis (MST), Surface Plasmon Resonance (SPR), and / or Isothermal Titration Calorimetry (ITC). These techniques are known in the art and are utilized to investigate molecular interactions. Employing different methods enables the measurement of interaction formation, strength, and dynamics between molecules, thus offering comprehensive insights into their interactions.

[0155] In embodiments, the binding of the peptide or peptidomimetic thereof to the recombinase can be assessed by biophysical and / or biochemical detection methods. In embodiments, the interaction between the peptide or peptidomimetic thereof and the recombinase can be assessed FRET, FCS, EMSA, Co-IP, SPR, and / or ITC. In embodiments, the binding strength between the peptide or peptidomimetic thereof and the recombinase can be assessed FRET, FCS, EMSA, Co-IP, SPR, and / or ITC. In embodiments, the interaction dynamics between the peptide or peptidomimetic thereof and the recombinase can be assessed FRET, FCS, EMSA, Co-IP, MST, SPR, and / or ITC. “Antimicrobial therapy” involves using pharmaceutical agents to combat microbial infections, aiming to eradicate bacteria at the site of infection. It is commonly employed for various infectious diseases, such as osteomyelitis, endocarditis, cystic fibrosis, tuberculosis, sinusitis, skin infections, ear infections, Lyme disease, urinary tract infections, and / or prosthetic joint infection; and / or for immunocompromised medical conditions; and / or before and / or after surgery. However, in some cases, it is used prophylactically. Evaluating antimicrobial agents typically involves preclinical studies assessing in vitro and in vivo efficacy, followed by clinical trials focusing on safety and clinical outcomes. Antimicrobial therapy aims to achieve bacterial eradication, although clinical trials often prioritize demonstrating equivalence rather than eradication. Pharmacokinetic and pharmacodynamic analyses are essential for predicting bacteriologic efficacy and guiding dose recommendations. Maximizing bacterial eradication enhances clinical outcomes and mitigates the emergence and spread of resistant pathogens. The prudent use of antimicrobial therapy involves accurate diagnosis, understanding drug characteristics, considering host factors, and adhering to antimicrobial stewardship principles to ensure responsible use.

[0156] In embodiments, the present invention relates to the peptide or peptidomimetic for use in the treatment of patients in need of antimicrobial therapy.

[0157] In embodiments, the peptide or peptidomimetic thereof is intended for use according to the invention, wherein the patient a. has a chronic infection, preferably osteomyelitis, endocarditis, cystic fibrosis, tuberculosis, sinusitis, skin infections, ear infections, Lyme disease, urinary tract infections, and / or prosthetic joint infection; and / or b. has an immunocompromised medical condition; and / or c. will undergo or has undergone a surgery.

[0158] The terms “disorder,” “disease,” or “medical condition” as used herein, can be used interchangeably.

[0159] The terms “chronic condition,” “chronic disease,” or “chronical medical impairment” are used interchangeably and are characterized by its persistent or long-lasting effects, typically lasting over three months. Chronic conditions often impact multiple areas of the body, exhibit incomplete responsiveness to treatment, and endure over an extended duration. Periods of remission and relapse, where the condition temporarily improves or reoccurs, are common in chronic diseases. These conditions are frequently linked to non-communicable diseases, which have non-infectious causes and encompass a range of health states, including syndromes, physical impairments, disabilities, and diseases.

[0160] “Immunodeficiency” refers to a condition where the immune system is weakened or compromised, leading to an increased susceptibility to infections and other diseases. This can be caused by various factors, including certain diseases like HIV / AIDS, cancer, and diabetes, as well as medications such as chemotherapy and immunosuppressive drugs used in organ transplants. Immunocompromised individuals may be more prone to opportunistic infections and may have reduced vaccine responses. There are two main categories of immunodeficiency: primary, which is inherited and present from birth, and secondary, which is acquired later in life due to external factors like medications, chronic illnesses, or environmental toxins. Primary immunodeficiencies include a - T1 - range of genetic disorders affecting different components of the immune system, while secondary immunodeficiencies can result from conditions like malnutrition, aging, certain medications, and chronic infections. Additionally, autoimmune diseases, hormonal disorders, and lifestyle factors like smoking and alcoholism can also contribute to immunodeficiency.

[0161] In one embodiment, the peptide or peptidomimetic is used prophylactically.

[0162] “Antibiotics” or antibacterials, are a category of antimicrobial substances specifically targeting bacteria. Widely used in medical practice, antibiotics play a crucial role in treating and preventing bacterial infections by either killing bacteria or inhibiting their growth. While some antibiotics may also exhibit activity against protozoa, they are ineffective against viral and fungal infections. The term "antibiotics" is often used interchangeably with "antibacterials," encompassing both naturally produced antibiotics and fully synthetic antibacterial agents. Medical usage typically reserves "antibiotics" for naturally derived substances, while fully synthetic compounds fall under "nonantibiotic antibacterials." Antibiotics are prescribed based on empiric therapy when the infecting pathogen is unidentified and definitive therapy when the pathogen is known. They are also utilized preventively in at-risk populations and surgical procedures to prevent infections. Antibiotics are classified based on their mechanism of action, chemical structure, or spectrum of activity, with most targeting bacterial functions or growth processes.

[0163] “Antibiotic resistance,” a subset of antimicrobial resistance, refers to the ability of bacteria to withstand the effects of antibiotics that were previously effective in treating infections caused by those bacteria. Individuals do not acquire this resistance; instead, it is inherent to the microbes, resulting from genetic mutations or the acquisition of resistance genes. Horizontal gene transfer, facilitated by mechanisms like conjugation, transduction, or transformation, enables the spread of resistance genes among bacterial populations. The widespread prevalence of antibiotic-resistant bacteria poses a significant global health threat, leading to increased mortality rates and rendering previously effective antibiotics less potent. Bacteria develop resistance to antibiotics through various mechanisms. Enzymes like p-lactamases can chemically modify antibiotics, rendering them ineffective, while bacteria may alter their cellular targets to prevent antibiotic binding, as seen in MRSA (Methicillin-Resistant Staphylococcus Aureus). They may also bypass inhibited metabolic pathways or reduce drug accumulation by decreasing permeability or actively pumping drugs. Bacteria can utilize heat shock proteins to continue protein synthesis stalled by antibiotics. Resistance mechanisms can be intrinsic or acquired through mutations or the acquisition of extra- chromosomal DNA, such as plasmids. Horizontal gene transfer disseminates resistance genes among bacterial populations, contributing to widespread antibiotic resistance. These mechanisms underscore bacteria's adaptability and pose challenges in combating antibiotic resistance effectively.

[0164] In embodiments, the peptide or peptidomimetic reduces antibiotic resistance. In embodiments, the peptide or peptidomimetic reduces antimicrobial resistance. In embodiments, the peptide or peptidomimetic reduces spread of resistance genes among bacterial populations.

[0165] Different antibiotic resistance genes (ARGs) of bacteria can develop resistance to different types of antibiotics. Resistance genes can be divided into the following categories based on the class of antibiotics they grant resistance to which include tetracyclines (tef), sulfonamides (st / / ) , p-lactams (b / a), macrolides (erm), aminoglycosides (aac), fluoroquinolone (fca), colistin (mcr), vancomycin (van), and multidrug (mdr). The intracellular ARGs (iARGs) are a major component of ARGs in nutrient-rich environments, while the extracellular ARGs (eARGs) are prevalent in aquatic environments. The eARGs can be adsorbed by soil and sediment particles, avoid DNase degradation, and thus, persist longer than the iARGs. Taken together, these features underscore the critical role of the eARGs in the spread of antibiotic resistance in the environment. The most common ARGs from hospitals were multidrug, glycopeptide, and p-lactamide ARGs (mecA, vanA, vanB, and bla), while sulfonamide and tetracycline ARGs (sul and tef) were the most common ARGs from farms, wastewater treatment plants (WWTPs), water, and soil. In six types of habitats (farms, cities, WWTPs, water, soil, and air), the first 50 subtypes of ARGs included eight antibiotic families: P-lactam (bla), sulfanilamide (suf), tetracycline (tef), aminoglycoside (aad), multidrug (mec), amphenicol (flo), trimethopolyl (dfr), and glycopeptide (van). Among them, mecA (multidrug) was the most common in the world, followed by -lactam (bla), glycopeptides (vanA, vanB), sulfonamide (sul1, sul2), and tetracycline (tetM). The top 10 ARGs reported from Asia included blaNDM-i, blacrx-M- 15, mecA, blarEM-1, sul1, vanA, blaKPc-2, sul2, blacrx-M- , and blaoxA-48 (responsible for -lactam, multidrug, sulfonamide, and glycopeptide antibiotics).

[0166] In embodiments, the peptide or peptidomimetic prevents antibiotic resistance genes from breaking down antibiotics. In embodiments, the peptide or peptidomimetic prevents the transfer of antibiotic resistance genes between bacteria. In embodiments, the peptide or peptidomimetic prevents antibiotic resistance gene transfer after conjugation. In embodiments, the peptide or peptidomimetic prevents antibiotic resistance gene transfer through plasmids.

[0167] In embodiments, the peptide or peptidomimetic prevents and / or slows down the spread of antibiotic resistance genes.

[0168] In embodiments, the antibiotic resistance genes are selected from the group of tetracyclines (tef), sulfonamides (suf), p-lactams (bla), macrolides (erm), aminoglycosides (aac), fluoroquinolone (fca), colistin (mcr), vancomycin (van), and / or multidrug (mdr).

[0169] As used herein, the terms "patient,” “individual," or "subject" are often used interchangeably and refer to any animal or human that exhibits a symptom of a disease, disorder, or condition that can be treated with the peptide or peptidomimetic and methods disclosed herein.

[0170] As used herein, the terms “(medical) disease,” “(medical) disorder,” and “(medical) condition” are often used interchangeably.

[0171] As used herein, "treatment" or "treating" includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can optionally involve either the reduction or amelioration of symptoms of the disease or condition or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease, condition, or associated symptoms. The phrase “therapeutically effective” is intended to include, within the scope of sound medical judgment, excessive toxicity, irritation, and / or other problems or complications but commensurate with a reasonable benefit / risk ratio.

[0172] As used herein, "prevent" and similar words such as "prevented," "preventing," or "prophylactic," etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effect, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0173] In one aspect, the present invention relates to a peptide or peptidomimetic for use in the treatment of patients in need of antimicrobial therapy.

[0174] In embodiments, the peptide or peptidomimetic for use in the treatment of patients in need of antimicrobial therapy comprises administering an effective amount, e.g., a therapeutically effective amount of the peptide or peptidomimetic contemplated herein. The quantity and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although clinical trials may determine appropriate dosages.

[0175] In embodiments, the peptide or peptidomimetic can be administered in combination, or simultaneously, with an antibiotic. As used herein, combined administration encompasses simultaneous treatment, co-treatment or joint treatment, and includes the administration of separate formulations of the peptide or peptidomimetic with an antibiotic, whereby treatment may occur in the same dosage form, within minutes of each other, in the same hour, on the same day, in the same week or in the same month as one another. Sequential administration of any given combination of combined agents is also encompassed by the term “combined administration”. A combination medicament, comprising the peptide or peptidomimetic with an antibiotic, may also be used in order to co-administer the various components in a single administration or dosage.

[0176] Pharmaceutical compositions comprising the peptide or peptidomimetic for administration to a subject can include, in embodiments, at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to the molecule of choice. Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional. The person skilled in the art is aware of compositions and formulations suitable for pharmaceutical delivery of the bi-specific agent disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

[0177] The peptide or peptidomimetic can, in embodiments, be combined with pharmaceutically acceptable carrier substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions comprising the peptide or peptidomimetic, conventional non-toxic pharmaceutically acceptable vehicles can be used, which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutical compositions, whether they are solutions, suspensions, or other like forms, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0178] A peptide or peptidomimetic comprised in a (pharmaceutical) composition can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intraocular, intranasal, intrapulmonary, or transdermal delivery, intramuscular, intraocular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal, intracerebroventricular, or parenteral routes.

[0179] In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of peptide or peptidomimetic may, in embodiments, be administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and / or ameliorate a selected condition or one or more symptom(s) thereof.

[0180] The attending clinician can vary dosage to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.

[0181] The instant disclosure also includes kits, packages, and multi-container units containing the herein- described peptide or peptidomimetic or pharmaceutical compositions comprising the same and / or means for administering the same for use in the prevention and treatment of conditions described herein and other conditions in human subjects.

[0182] All words and terms used herein shall have the same meaning commonly given to them by the person skilled in the art unless the context indicates a different meaning. All terms used in the singular shall include the plural of that term and vice versa.

[0183] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain, using most routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification indicate the skill level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. However, the disclosure supports a definition of only alternatives and "and / or." Throughout this application, where relevant, the term "about" indicates that a value includes the inherent variation of error for the device, the method employed to determine the value or the variation among the study subjects.

[0184] FIGURES

[0185] The invention is demonstrated by way of example in the following figures. The figures are to provide a further description of potentially preferred embodiments that enhance the support of one or more non-limiting embodiments of the invention.

[0186] Brief description of the figures:

[0187] Figure 1 : Schematic of bacterial integron.

[0188] Figure 2: Structural representation of the peptide (green) in the binding pocket.

[0189] Figure 3: Synapse architecture of VchlntlA I atCvcRbs.

[0190] Figure 4: Stability of the synaptic complex.

[0191] Figure 5: Stabilizing protein-protein interactions in the synaptic complex.

[0192] Figure 6: ConSurf results for homologous integrase I ntl4.

[0193] Figure 7: Stabilizing protein-protein interactions in the synaptic complex.

[0194] Figure 8: Destabilization of the Inti 1 :attCaadA7bssynaptic complex in the presence of 10 pM pL or pS.

[0195] Figure 9: Cross-class destabilization of the lntl4:attCaadA7bssynaptic complex in the presence of 10 pM pS4 or pS.

[0196] Figure 10: Sequence alignment of recombinases.

[0197] Figure 11 : Proposed mechanism for recombination efficiency modulation via synaptic complex stability

[0198] Detailed description of the figures:

[0199] Figure 1 : Schematic of Bacterial integron. (A) Regulatory mechanism of the integron with Inti and gene cassettes flanked by attl and attC sites. (B) Recombination cycle with excision of cassettes between two attC sites and insertion with attC / attl sites.

[0200] Figure 2: Structural representation of the peptide (green) in the binding pocket. The different colors of the binding pocket reflect different stretches of amino acids along the primary sequence, i.e. several distant stretches along the primary sequence for the final binding pocket.

[0201] Figure 3: Synapse architecture of VchlntlA / atCvcRbs. (A) Synaptic complex with active integrase subunits colored orange, inactive - blue. Folded attC sites are depicted in green and violet. The conserved 5’-AAC-3’ triplet is indicated in red as well as the attacking tyrosine. (B) A zoomed-in view on relevant protein-DNA interactions in cis and in trans. EHBs interactions are indicated along with involved protein structures. PDB ID: 2A3V. Visualized with PyMOL (Schrodinger, L., & DeLano, W. 2020)

[0202] Figure 4: Stability of the synaptic complex. (A) Development of a new DNA construct with two attC sites and a long ssDNA spacer to probe synaptic complex stability. After complex formation (1), the force is increased until disassembly (2) and further increase to displace all Integrase monomers (3). Force reduction leads to new synaptic complex formation (1) and the experiment can be repeated. (B) Correlation of mechanical stability and recombination efficiencies for various attC sites, suggesting a allosteric regulation of complex stability by DNA substructures. (C) EMSA of double- attC construct showing I ntl 1 dependent binding of up to 4 I ntl 1 monomers Vorobevskaia et al. 2024.

[0203] Figure 5: Stabilizing protein -protein interactions in the synaptic complex. (A) Structural model of the synaptic complex of Intl4 with two attC sites (PDB ID 2A3V). Active subunits (orange) and inactive subunits (blue) are highlighted. (B) Visualization of the binding pocket interactions to the short C-terminal a-helix between adjacent attCs and on the same attC hairpin. (C) AlphaFold2 model of the Inti 1 including the terminal a-helix and comparison to crystal structure of Intl4. Visualized with PyMOL (Schrodinger, L., & DeLano, W. 2020). (D) Sequence alignment of Intl4 and Inti 1 .

[0204] Figure 6: ConSurf results for homologous integrase Intl4. (A) The amino acid sequence of the I ntl4 with highlighted residues depending on their predicted conservation value, including the scale. (B) Visualization of the binding pocket interactions to the C-terminal a-helix for two binding modes. The residues of the pocket are color-coded according to the conservation scale. Visualized with PyMOL (Schrodinger, L., & DeLano, W. 2020)

[0205] Figure 7: Stabilizing protein -protein interactions in the synaptic complex. (A) Synaptic complex disassembly forces of protein mutants and (B) Inti 1 unbinding forces of full length and mutant proteins. (C) Destabilization of the synaptic complex of Inti 1 full length in presence of 10 pM of npS or npL.

[0206] Figure 8: Destabilization of the lntl1 :attCaadA7bssynaptic complex in the presence of 10 pM pL or pS. (A) Boxplots of synapse disassembly force for Inti 1 :attCaadA7bsin the presence of 1 % DMSO, pL, and pS; as well as Inti 1AC:attCaadA7bs. (B) Boxplots of synapse disassembly force for IntH :attCaadA7bsin the presence of different concentrations of pS (2 pM, 5 pM, 10 pM and 50 pM); as well as two pS variants: pS_v1 and pS_v2 (10 pM).

[0207] Figure 9: Cross-class destabilization of the lntl4:attCaadA7bssynaptic complex in the presence of 10 pM pS4 or pS. Boxplots of synapse disassembly force for lntl4:attCaadA7bswithout peptide and in the presence of 10 pM of pS4 or pS.

[0208] Fig. 10: Sequence alignment of recombinases. The sequence alignment of distinct recombinases show (partial) conservation. Figure 11 : Proposed mechanism for recombination efficiency modulation via synaptic complex stability. The proposed model of the interplay between the variable synaptic complex stability and the cellular processes that introduce tensile stress on the DNA in vivo.

[0209] EXAMPLES

[0210] The invention is demonstrated by way of the examples disclosed below. The examples provide technical support for and a more detailed description of potentially preferred, non-limiting embodiments of the invention.

[0211] As used herein, npS or pS refers to the “natural peptide short” with the sequence RSPLDAL; npL or pL refers to the “natural peptide long” with the sequence RSPLDALPPLTSER.

[0212] Summary of the Examples

[0213] In order to demonstrate the functionality and beneficial properties of the peptide and / or the peptidomimetic thereof described herein, the following examples are presented:

[0214] Synaptic complex destabilization by a small peptide

[0215] Modulation of the stability of the synaptic complex

[0216] Conservation of the binding pocket for the C-terminal a-helix

[0217] Destabilization of the synaptic complex by long and short peptides

[0218] Stabilizing protein-protein interactions in the synaptic complex

[0219] Recombination efficiency of integron is modulated by synaptic complex stability

[0220] Destabilization of the Inti 1 :attCaadA7bssynaptic complex in the presence of pS peptide or peptide variants

[0221] Cross-class destabilization of the lntl4:attCaadA7bssynaptic complex in the presence of 10 pM pS4 or pS.

[0222] Example 1: Synaptic complex destabilization by a small peptide

[0223] Our experiments demonstrate that recombination efficiency is correlated to the mechanical stability of the integron synaptic complex. By extension, destabilization of a given synapse formation reduces the overall recombination efficiency of the system, thus slowing down the rates of resistance growth in bacterial populations. So far, we have investigated the effects of different DNA substrates and protein variants and found that both can lead to a less stable synapse. We therefore develop a method to influence a given synaptic assembly regardless of its attC site or integrase variant involved. Eliminating C-terminal a-helix docking to the neighboring subunit in the Inti 1ACtetramer was shown to have a big destabilization impact. Therefore, we present that preventing C-terminal a- helix interaction in the Inti 1 tetramer by blocking the binding pockets of the subunits with a small molecule, reduces synaptic complex stability. Given known sequences of the C-terminal a-helix, we have designed peptides, which bind to the pocket of the integrase. We referred to the known crystallographic structure (MacDonald et al. 2006) of the homologous integrase Intl4, which ends with the a-helix and has no additional tail structure. Therefore, we decided to implement two designs in our peptide sequence and show how the unstructured end tail influences docking to the integrase monomers. One example peptide mimics the docking a-helix alone (peptide short - pS) and the other example is longer, containing also the unstructured amino acid chain that is present in our integrase Inti 1 (peptide long - pL).

[0224] Example 2: Modulation of the stability of the synaptic complex.

[0225] We have addressed the question of whether attC hairpins modulate the stability of the synaptic complex and which key amino acid residues and structural features of the integrase modulate the stability of the synaptic complex (Vorobevskaia et al., 2024). To address the stability of the synaptic complex, we have developed an advanced, highly sensitive assay based on single-molecule optical tweezer force spectroscopy. This study focused on the synaptic complex formed by two attC sites and an Inti 1 integrase. For this purpose, we developed a method to immobilize a long ssDNA (~ 300 nts) containing two desired attC sites between two DNA handles for subsequent optical tweezers experiments (Figure 4). The experiment was designed such that after synaptic complex formation (step 1), we can increase the destabilizing force on the synaptic complex until it breaks apart. A 100 nt long, unstructured ssDNA is exposed, and the tether length increases by a characteristic fingerprint of ~71 nm (step 2). Upon further increase of the force, the integrase monomers will disassemble, and we will expose the full ~ 300 nt ssDNA to the solution (step 3). After reducing the distance between both laser traps, the force is reduced, attC sites can fold again, a new synaptic complex will form, and the experiment can be repeated. Using this assay, plenty of information about synaptic complexes can be gathered, among which we analyze: (i) the synaptic complex disassembly force, (ii) the probability of forming a synaptic complex, (iii) the probability of forming other incomplete complexes or only hairpin bound states. Here, we focus on the synaptic complex disassembly force for different attC sites, showing recombination frequencies ranging from high (affCaadA7bs) to low (affCvcRinv) to very low for the synthetic attC site L2. We found that the synaptic complex disassembles on average at forces ranging from ~14 pN to ~7 pN. Noteworthy, RNA polymerases or DNA helicases typically generate forces on the order of 10-30 pN, sufficient to destabilize or disrupt a synaptic complex and cause abortion of integrase recombination. We have also observed a strong correlation between the recombination efficiency observed in vivo experiments and the synaptic complex disassembly force (Figure 4). Binding to the hairpins itself was still observable, also in electrophoretic mobility shift assays (Figure 4) for up to four integrases. Binding of four Inti 1 monomers does not directly report about the formation of a synaptic complex; this is measured in optical tweezers experiments with the dedicated fingerprint of a 71 nm contour length change upon synaptic complex breakdown; see above. The correlation between mechanical destabilization for different attC sites indicates a strong allosteric communication from attC to I ntl 1 and the synaptic complex.

[0226] Example 3: Conservation of the binding pocket for the C-terminal a-helix

[0227] To elucidate binding geometry of the proposed peptides, we investigated the position of the Intl4 terminal a-helix. As mentioned above, there are two types of helix-pocket interactions: in cis and in trans. Even though the a-helix occupies the same pocket on the neighboring subunit in both cases, the binding geometry and key interactions are slightly different (Figure 5). The first two amino acids of the a-helix are Ser315 and Pro316 (according to Intl4 resolved structure MacDonald et al. 2006)). Both these residues are involved in the docking to the binding pocket for both types of subunits (active and inactive). These two interaction modes are very similar to the terminal a-helix docks to the same region, but they happen at slightly different angles. The proline residue seems to occupy a cavity that is present for both in cis and in trans modes. However, no specific interactions are involved. Ser315 has a potential polar interaction of the sidechain OH to the backbone COOH of Gly131 ; the distance between the atoms is less than 3A, according to the PyMOL interaction identifier (Schrodinger, L., & DeLano, W. 2020). In the mode of in trans interaction, two terminal amino acids (Arg319 and Leu320) are positioned to allow polar interaction between their backbone COOH to the sidechains of Arg108 and Arg112. This might be the stabilization interaction to keep the end of the helix in the pocket. In cis mode, these interactions are lacking due to the helix orientation, as both these residues face outside the pocket. However, in cis mode, Ser318 can interact with the sidechain of Glu172, adding stability to the helical docking.

[0228] To access the potential binding effects of mimicking peptides on other integrase types, we used ConSurf (Ashkenazy et al. 2016) to analyze crystallographically resolved Intl4 integrase in terms of the terminal helix conservation (Figure 6). Most of the amino acids that constitute the helix are conserved. Establishing the beginning of the a-helical structure and being very conserved, Ser315 and Pro316 might be involved in the starting recognition phase or the stabilization of the docking. Ser318 is also highly conserved and can potentially play a stabilizing role in cis mode of helical docking.

[0229] The high level of conservation and moderate sequence similarity of the C-terminal a-helix between Intl4 and Inti 1 suggests that the terminal a-helical docking mechanism of inti 1 would also be similar. Noteworthy, C-terminal conservation was characteristic of other integron integrases and other members of the tyrosine recombinase family. For instance, a high similarity was identified for E. coli recombinases XerD, XerC, and an integrase from a bacteriophage HP1 . These site-specific recombinases shared the short terminal SPLS sequence (Ashkenazy et al. 2016; Subramanya et al. 1997; Castillo, Benmohamed, and Szatmari 2017; Hickman et al. 1997) and also form synaptic complexes, although they use double-stranded DNA substrates. Based on the available crystallographic structures, many tyrosine recombinases use the C-terminal docking interaction to connect the subunits of the assembly (Van Duyne 2007; Chen et al. 2000; Subramanya et al. 1997; Gopaul, Guo, and Van Duyne 1998). This general resemblance of the C-terminal a-helix function implies some evolutionary-determined interaction mode that adds synaptic complex stability and might act to promote the overall effectiveness of the system.

[0230] Example 4: Destabilization of the synaptic complex by long and short peptides

[0231] The peptides were synthesized, and their effects were probed using the single-molecule optical tweezers stability assay with the reference attCaadA7bs and full-length Inti 1 protein. Therefore, the introduced peptides had the amino acid sequences of Inti 1 C-terminal region: the a-helix alone (pS) and together with the unstructured terminal tail (pL).

[0232] To potentially saturate the integrase binding site, we incubated IntH with either the long or the short peptide versions at a peptide concentration of 10 pM. Control measurement of the lntl1 :a#CaacM7bs complex without the peptide was taken for every formed specific tether prior to the exposure to the incubated Inti 1 :peptide solution. We found that integrase incubated with the designed long peptide still formed the synaptic complex that demonstrated lower stability. The mean disassembly force of the synapse:pL complex was reduced to 10.2 pN ± 0.5 pN (N = 178, mean ± SEM), which is lower than demonstrated for the alanine mutant Inti 1 A, but higher than was achieved using the C-terminal deletion integrase IntHAC(diss = 6.8 pN). Interestingly, the shorter peptide, which consists of only seven amino acids, showed the same effect as the long one (Figure 8). The mean disassembly force of the synapse in the presence of the short peptide (pS) was reduced to 10.4 pN ± 0.4 pN (N = 221 , mean ± SEM).

[0233] The initial peptide stock was dissolved in DMSO. Therefore, our final peptide-protein sample had a remaining 1% DMSO. To investigate the possible influence of DMSO on integrase and synaptic complex assembly and stability, we performed additional control experiments without the peptide but with 1% DMSO in the integrase solution and incubated for 30 minutes prior to the measurements replicating the preparation procedure used with peptides. We gathered a negative control dataset of comparable size to our reference Inti 1 .attCaadm (171 synapse disassembly traces versus 169 traces, respectively). The disassembly force distribution resembles our initial IntH .attCaadA7bs distribution (diss = 14.6 pN ± 0.7 pN (mean ± SEM), versus 13.9 pN), and according to the Welch T- test (Freedman, Pisani, and Purves 2007) and the Kolmogorov-Smirnov test no significant difference was found. This control supports our interpretation that pS and pL destabilize the synaptic complex. Noteworthy, the 1 % DMSO experiment can be interpreted as the technical replicate for the reference Inti 1 attCaadA7bs dataset, confirming our initial dataset on its mechanical stability.

[0234] Example 5: Stabilizing protein-protein interactions in the synaptic complex.

[0235] The 3D structure of Inti 1 has not been solved; however, as mentioned above, the structure of Intl4 has been solved by X-ray crystallography at 2.8 A (PDBId 2a3v). In this structure, following a highly flexible loop, the C-terminal a-helix of one protein monomer docks in a small C-terminal binding pocket (CtBP) of the neighboring subunit leading to a circular interlocking connection among the four monomers of the tetrameric complex, a feature also appreciated in other Tyr-recombinases Visual inspection of the interactions established by each of the four a-helices within their corresponding CtBPs in the contiguous monomer (Figure 5) shows a slight difference depending on if it is an active subunit docking its C-terminus into an inactive subunit or vice versa, suggesting some - minor but potentially regulatory - allosteric changes. We subsequently analyzed conservations of the Intl4 surface using the ConSurf server (Figure 6). Among the candidates provided by the web server, we found non-surprisingly the Intl4 homolog Inti 1 , which we study, with a high conservation of many residues and, in particular, those central in the sequence of the C-terminal a-helix (i.e.315SPLS; residue numbering according to I ntl4 structure; Figure 6). An AlphaFold prediction with the Google Colab server (ColabFold v1 ,5.2-patch: AlphaFold2) of the Inti 1 structure also depicted the C- terminus consisting of a largely unstructured region, like in the structure of Intl4, followed by an a- helix (Figures. Noteworthy, the ConSurf web server also found an evolutionary high conservation of the C-terminus in (i) the E. coli recombinase XerD, which is part of the Xer recombination system, and (ii) the bacteriophage HP1 integrase, which integrates the HP1 genome into Haemophilus influenzae. These findings suggest a general evolutionary CtBP interaction site, which might stabilize many synaptic complexes. Apart from the prominent cyclic C-terminal tail interaction described above we have identified synaptic complex-specific protein-DNA interactions including the 12 helix of the Intl4 making contact to the adjacent attC site in trans that might play an important role, as suggested by the structural study. We generated two Inti 1 variants, where we have (i) mutated K219A and K220A in the I2 helix (IntHl2A), which are in contact with extra-helical bases of the adjacent attC site, and (ii) truncated the C-terminal tail (A321stop) cutting of the small a-helix (Inti 1AC). For both Inti 1 variants, our preliminary results show that the synaptic complex disassembly force was strongly reduced (~ 11 pN and 7 pN, respectively), compared to the I ntl 1 , indicating that the contact of I2 to DNA and the C-terminal interlocking are critical to form a stable synaptic complex (Figure 7A). Note, the unbinding forces of the IntH , lntl1l2A, and Inti 1DCfrom attC hairpins were nearly unchanged, indicating that binding to DNA itself was not affected by our mutations (Figure 7B).

[0236] Having gained a basic understanding that recombination efficiency correlates with the stability of the synaptic complex and that mutations within Inti 1 affect synaptic complex stability, we became interested in establishing new strategies to destabilize the synaptic complex and, by extension, potentially reduce the recombination efficiency. The interaction of I2 with DNA is deep buried within the synaptic complex; thus, it appears less likely to be reached by, e.g., small molecules. Therefore, we have focused on the interlocking interaction via the C-terminal docking to the CtBP. We hypothesized that blocking of the CtBP might represent a promising target. We, therefore, decided to test whether two peptides resembling the C-terminal sequence of Inti 1 (i.e., natural peptide Long (npL) and Short (npS) of 14 and 7 amino acids length, respectively; Figure 5C) could compete to occupy the CtBP and prevent the C-terminal helix from binding. In preliminary work, we have repeated our experiments with Inti 1 wildtype and two attCaadA7 sites in the absence of those peptides and the presence of either of them at 10 pM concentration. In the absence of the peptides, we have corroborated our earlier results and found a synaptic complex disassembly force of ~ 14 pN. In the presence of the npL and npS peptides, the mean disassembly force drops below 10 pN, showing a strong destabilization of the synaptic complex (Figure 9). Both molecules are anticipated to lay ground for the development of an efficient inhibition of recombination and adaptation driven by IntH .

[0237] Example 6. Recombination efficiency of integron is modulated by synaptic complex stability

[0238] We plotted the relative recombination efficiency of the cassette excision versus the mean synaptic complex disassembly force (a measure of the synaptic complex stability) and find a clear correlation between both parameters strongly suggesting that the mechanical stability modules the recombination efficiency (Figure 10). Subsequently, we plotted literature values of the relative recombination efficiencies originating from integration (attC x attl) complexes, which also follow the correlation trend. We did not observe mean disassembly forces below 6 pN in any of our DNA- protein combinations, indicating that the system has reached a low threshold of sustainable stability. We propose that the threshold correlates with the average tensile forces that act on the DNA in vivo during multiple cellular processes, such as translation, transcription, or replication (Michelle D. Wang et al. 1998; Yodh, Schlierf, and Ha 2010; Goel et al. 2001 ; Thomen, Lopez, and Heslot 2005). Mechanically weak complexes will have a very short lifetime due to the exponentially accelerated disassembly kinetics with force, effectively inhibiting successful completion of recombination.

[0239] Example 7: Destabilization of the IntH :attCaadA7bssynaptic complex in the presence ofpS peptide or peptide variants. We observe the destabilization effect correlating with the increasing concentration of the pS peptide (Fig. 8). In the presence of 2 pM pS, the synaptic complex disassembly force is unchanged from the reference IntH :a#Caa<M7bsmeasurements without peptide. However, upon peptide concentration increase, we observe the destabilization of the synaptic complex, which becomes most prominent at 10 pM pS concentration and does not improve at 50 pM pS, indicating that perhaps the complex is fully saturated with the peptide at 10 pM pS. As the destabilization effect is clearly concentrationdependent, it supports our idea of the specific peptide-integrase interaction.

[0240] We have also tested two pS variants that differ from the pS peptide in one amino acid each. pS_v2 has (Ser2->Ala substitution) with the following sequence: RAPLDAL and pS_v1 has (Leu4->Ala substitution) with the following sequence: RSPADAL. The S2A substitution abolished the putative polar interaction between S2 and the binding pocket and led to no destabilization effect as expected. However, the L4A substitution does not seem to affect the peptide’s destabilization activity. It might be because Alanine has very similar biochemical parameters to Leucine and does not disturb the structure or the putative hydrophobic interactions upon binding.

[0241] Example 8: Cross-class destabilization of the lntl4:attCaadA7bssynaptic complex in the presence of 10 pM pS4 or pS.

[0242] We have tested the peptide destabilization on another class of integron integrase - I ntl4 (Fig. 9). We observed destabilization effects in the presence of the pS peptide, which has a different sequence with respect to the C-terminal tail sequence of I ntl4. Therefore, we have shown that the peptide derived from the class 1 integrase destabilizes the class 4 integrase. This cross-class activity supports the idea of utilizing one compound, disregarding what class of the integrase is present for the synaptic complex destabilization. We have also tested the effect of the pS4 peptide (derived from the sequence of the Intl4 C-terminal tail) on the synaptic complex stability for lntl4:a#Caa<M7bsand observed similar destabilization activity.

[0243] Tests are ongoing, for example using the above-mentioned assays, for additional peptide sequences according to

[0244] • pS_v3 (R1 S): SSPLDAL

[0245] • pS_v4 (R1 L): LSPLDAL

[0246] • pS_v5 (D5S): RSPLSAL

[0247] • pS_v6 (A7R): RSPLDRL

[0248] Conclusion of the Examples

[0249] We are able to show a correlation between different recombination efficiency values demonstrated by the integron system in vivo and the mechanical stability of its crucial formation - the synaptic complex. We established that a destabilized synapse leads to reduced recombination efficiency and can be achieved in particular, via protein mutation. The most prominent destabilization effect we observed was characteristic of the Inti 1ACwhere we discovered that the C-terminal loop-like interaction between subunits greatly promotes the formation’s stability. We replicated the destabilization effect on the full-length integrase by using a competitor molecule that would occupy the binding site of the C-terminal tail and prevent the latter from docking effectively inhibiting the stabilizing protein-protein interaction. We used two peptide molecules of different lengths that mimic either the full sequence of the Inti 1 C-terminal tail (pL) or only the part of it and are comprised of the small helical structure (pS). Our designed novel peptide molecules indeed demonstrated considerable destabilization effects already at 10 pM concentration on a given synapse formation. We have thus developed a novel approach for potential recombination efficiency reduction through synaptic complex destabilization. The designed interference molecules show clinical relevance as they can be used as a supplement, together with an antimicrobial therapy, reducing recombination efficiency of the integron system and slowing the rates of multiple antibiotic resistances in pathogenic bacteria.

[0250] REFERENCES

[0251] 1. MacDonald, D., Demarre, G., Bouvier, M., Mazel, D., and Gopaul, D.N. (2006). Structural basis for broad DNA-specificity in integron recombination. Nature 440, 1157-1162. 10.1038 / nature04643.

[0252] 2. Demarre, G., Frumerie, C., Gopaul, D.N., and Mazel, D. (2007). Identification of key structural determinants of the IntH integron integrase that influence attCx attll recombination efficiency. Nucleic acids research 35, 6475-6489.

[0253] 3. Van Duyne, G.D. (2007). A Structural View of Tyrosine Recombinase Site-Specific Recombination. In Mobile DNA II (eds N.L. Craig, R. Craigie, M. Gellert and A.M. Lambowitz).

[0254] 4. Meinke, G., Bohm, A., Hauber, J., Pisabarro, M.T., and Buchholz, F. (2016). Cre Recombinase and Other Tyrosine Recombinases. Chem. Rev. 116, 12785-12820. 10.1021 / acs.chemrev.6b00077.

[0255] 5. Gibb B, Gupta K, Ghosh K, Sharp R, Chen J, Van Duyne GD. Requirements for catalysis in the Cre recombinase active site. Nucleic Acids Res. 2010 Sep;38(17):5817-32. doi: 10.1093 / nar / gkq384. Epub 2010 May 12. PMID: 20462863; PMCID: PMC2943603.

[0256] 6. Castillo F, Benmohamed A, Szatmari G. Xer Site Specific Recombination: Double and Single Recombinase Systems. Front Microbiol. 2017 Mar 20;8:453. doi: 10.3389 / fmicb.2017.00453. PMID: 28373867; PMCID: PMC5357621.

[0257] 7. Subramanya, H.S., Arciszewska, L.K., Baker, R.A., Bird, L.E., Sherratt, D.J., and Wigley, D.B. (1997). Crystal structure of the site-specific recombinase, XerD. The EMBO Journal 16, 5178-5187. 10.1093 / emboj / 16.17.5178.

[0258] 8. Chen Y, Narendra U, lype LE, Cox MM, Rice PA. Crystal structure of a Flp recombinase- Holliday junction complex: assembly of an active oligomer by helix swapping. Mol Cell. 2000 Oct;6(4):885-97. PMID: 11090626.

[0259] 9. Esposito D, Scocca JJ. The integrase family of tyrosine recombinases: evolution of a conserved active site domain. Nucleic Acids Res. 1997 Sep 15;25(18):3605-14. doi: 10.1093 / nar / 25.18.3605. PMID: 9278480; PMCID: PMC146934.

[0260] 10. Hickman, A.B., Waninger, S., Scocca, J. J., and Dyda, F. (1997). Molecular Organization in Site-Specific Recombination: The Catalytic Domain of Bacteriophage HP1 Integrase at 2.7 A Resolution. Cell 89, 227-237. 10.1016 / S0092-8674(00)80202-0.

[0261] 11. Ashkenazy, H., Abadi, S., Martz, E., Chay, O., Mayrose, I., Pupko, T., and Ben-Tai, N. (2016). ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Research 44, W344-W350. 10.1093 / nar / gkw408.

[0262] 12. Ip, Stephen C.Y., Migena Bregu, Frangois-Xavier Barre, and David J. Sherratt. (2003). Decatenation of DNA Circles by FtsK-Dependent Xer Site-Specific Recombination. The EMBO Journal 22 (23): 6399-6407. https: / / doi.org / 10.1093 / emboj / cdg589

[0263] 13. Landy, Arthur. 2015. “The A Integrase Site-Specific Recombination Pathway.” In Mobile DNA III, 91-118. John Wiley & Sons, Ltd. https: / / doi.org / 10.1128 / 9781555819217.ch4. Kwon, Hyock Joo, Radhakrishna Tirumalai, Arthur Landy, and Tom Ellenberger. 1997. “Flexibility in DNA Recombination: Structure of the Lambda Integrase Catalytic Core.” Science 276 (5309): 126-31. Jayaram, Makkuni, Chien-Hui Ma, Aashiq H Kachroo, Paul A Rowley, Piotr Guga, Hsui- Fang Fan, and Yuri Voziyanov. 2015. “An Overview of Tyrosine Site-Specific Recombination: From an Flp Perspective.” Microbiology Spectrum 3 (4): 10.1128 / microbiolspec.mdna3- 0021-2014. https: / / doi.Org / 10.1128 / microbiolspec.mdna3- 0021-2014 Schrodinger, L., & DeLano, W. 2020. “PyMOL.” http: / / www.pymol.org / pymol. Chen, Yu, Umadevi Narendra, Lisa E. lype, Michael M. Cox, and Phoebe A. Rice. 2000. "Crystal Structure of a Flp Recombinase-Holliday Junction Complex: Assembly of an Active Oligomer by Helix Swapping." Molecular Cell 6 (4): 885-97. https: / / doi.org / 10.1016 / S1097- 2765(05)00088-2. Gopaul, D. N., F. Guo, and G. D. Van Duyne. 1998. "Structure of the Holliday Junction Intermediate in Cre-LoxP Site-Specific Recombination." The EMBO Journal 17 (14): 4175- 87. https: / / d0i.0rg / l 0.1093 / emboj / 17.14.4175. Wang, Michelle D., Mark J. Schnitzer, Hong Yin, Robert Landick, Jeff Gelles, and Steven M. Block. 1998. “Force and Velocity Measured for Single Molecules of RNA Polymerase.” Science 282 (5390): 902-7. https: / / doi.org / 10.1126 / science.282.5390.902. Yodh, Jaya G., Michael Schlierf, and Taekjip Ha. 2010. “Insight into Helicase Mechanism and Function Revealed through Single-Molecule Approaches.” Quarterly Reviews of Biophysics 43 (2): 185-217. https: / / doi.org / 10.1017 / S0033583510000107. A. Goel, M. D. Frank-Kamenetskii, T. Ellenberger, D. Herschbach, Tuning DNA “strings”: Modulating the rate of DNA replication with mechanical tension. Proceedings of the National Academy of Sciences 98, 8485-8489 (2001) P. Thomen, P. J. Lopez, F. Heslot, Unravelling the Mechanism of RNA-Polymerase Forward Motion by Using Mechanical Force. Phys. Rev. Lett. 94, 128102 (2005) Vorobevskaia, Ekaterina, Celine Loot, Didier Mazel, and Michael Schlierf. 2024. “The Recombination Efficiency of the Bacterial Integron Depends on the Mechanical Stability of the Synaptic Complex.” Science Advances 10 (50): eadp8756. https: / / doi.Org / 10.1126 / sciadv.adp8756.

Claims

CLAIMS1 . A peptide comprising an amino acid sequence according toX1SPLX2X3L (SEQ ID NO 1), wherein Xi, X2, and X8are any amino acid, or a peptidomimetic thereof, that binds a target site of a recombinase, wherein the peptide or peptidomimetic comprises an alpha helix-like structure and has a length of 7 to 15 amino acids.

2. The peptide or the peptidomimetic thereof according to the preceding claim, wherein the peptide or peptidomimetic comprises an amino acid sequence according toX1SPLX2X3L (SEQ ID NO 1), wherein X R, K, Q, N, S, T, L; X2: D, E, N, S; and X3: A, V, L, I, R, E, G.

3. The peptide or the peptidomimetic thereof according to the preceding claim, wherein the peptide or peptidomimetic comprises an amino acid sequence according toX1SPLX2X3L (SEQ ID NO 1), wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G.

4. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or peptidomimetic comprises an amino acid sequence according to (SEQ ID NO. 2) RSPLDAL or a sequence with at least 70%, preferably 80%, sequence identity thereto.

5. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or peptidomimetic comprises an amino acid sequence according to (SEQ ID NO. 2) RSPLDAL.

6. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or peptidomimetic comprises an amino acid sequence according to(SEQ ID NO. 3) X1SPLX2X31X4X5X5X7X3X9X10, wherein X R, K, Q, N, S, T, L; X2: D, E, N, S; X3: A, V, L, I, R, E, G; X4: P, A, M, R, L; X5: P, A, L, Q; X6: L, O, V, M, H, A, F; X7: T, S, I, L, R; X8: S, T, N; X9: E, D, Q, P, G; and X : R, K, Q, N, G.

7. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or peptidomimetic comprises an amino acid sequence according to(SEQ ID NO. 3) X1SP 1X2X31X4X5X5X7X3X9X10, wherein X R, S, T, L; X2: D, S; and X3: A, R, E, G; X4: P, A, M, R; X5: P, L, Q; X6: L, H, A; X7: S, I, L, R; X8: S, T, N; X9: E, Q, P, G; and X : R, G.

8. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDALPPLTSER (SEQ ID NO. 4) or a sequence with at least 70%, preferably 80%, more preferably at least 90% identity thereto.

9. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or peptidomimetic comprises an amino acid sequence according to RSPLDALPPLTSER (SEQ ID NO. 4).

10. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or the peptidomimetic thereof reduces activity and / or efficiency of the recombinase.11 . The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide or the peptidomimetic thereof inhibits activity and / or efficiency of the recombinase.

12. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide competes with a native ligand of the recombinase.

13. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the peptide destabilizes a synaptic complex of the recombinase.

14. The peptide or the peptidomimetic thereof according to the preceding claim, wherein synapse assembly is hindered by blocking a C-terminal binding pocket (CtBP) of the recombinase.

15. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the recombinase comprises an amino acid sequence according to(SEQ ID NO. 5) FATX1LLX2X3G, wherein X H, A; X2: E, R; X3: V, S or a sequence with at least 70%, preferably 80%, more preferably at least 90% identity thereto.

16. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the recombinase is a tyrosine recombinase or an integrase.

17. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the recombinase is a bacterial integrase.

18. The peptide or the peptidomimetic thereof according to any one of the preceding claims, wherein the recombinase is an Inti integrase, a phage lambda integrase, HP1 integrase, Bacteriophage P22 integrase, Bacteriophage PF1 integrase, a cyclic recombinase (Cre), Cre P1 or any directed evolution derivatives, XerC / D recombinase, XerH, XerS and XerA recombinases, or a Flp recombinase.

19. An in vitro method for modulating recombinase activity using the peptide or peptidomimetic thereof according to any one of claims 1 -18.

20. An in vitro method for modulating recombinase efficiency using the peptide or peptidomimetic thereof according to any one of claims 1-18.

21. A mixture or composition comprising a recombinase according to any one of claims 15-18 and the peptide or peptidomimetic according to any one of claims 1-18.

22. A peptide or peptidomimetic according to any one of claims 1-18 for use in the treatment of patients in need of antimicrobial therapy.

23. The peptide or peptidomimetic for use according to claim 22, wherein the patient a. has a chronic infection, preferably osteomyelitis, endocarditis, cystic fibrosis, tuberculosis, sinusitis, skin infections, ear infections, Lyme disease, urinary tract infections, and / or prosthetic joint infection; and / or b. has an immunocompromised medical condition; and / or c. will undergo or has undergone a surgery.

24. A pharmaceutical composition comprising the peptide or peptidomimetic according to claims 1-18 with a pharmaceutically acceptable carrier.

25. The pharmaceutical composition according to claim 24, wherein the pharmaceutical composition is administered to a patient in combination with an antibiotic.

26. The pharmaceutical composition according to claim 24 or 25, wherein the pharmaceutical composition reduces or reduces the risk of bacterial resistance to one or more antibiotics.

Images