Bacterial lon protease domains and their use in therapy

Abstract

This invention relates to novel compositions, particularly compositions comprising one or more domains of bacterial Lon protease or related peptides. These compositions can be used to treat various diseases, such as infections, especially bacterial infections and cancer.

Description

Technical Field

[0001] This invention relates to novel compositions, particularly compositions comprising one or more domains of bacterial Lon protease or related peptides. These compositions can be used to treat various diseases, such as infections, especially bacterial infections and cancer. Background Technology

[0002] MYC transcription factors are important therapeutic targets. However, due to the lack of specific MYC inhibitors, they remain "undruggable".

[0003] Transcriptional control is crucial for maintaining normal tissue function and optimizing defenses against common diseases such as infections and cancer. MYC transcription factors are often considered "master gene regulators," controlling expression in over 15% of the human genome. It has been noted that all key aspects of cell proliferation, cell growth, and tumorigenesis are actively regulated by C-MYC; in appropriate amounts, C-MYC is essential for normal cellular function, but its dysregulation is extremely dangerous. MYC dysregulation has been proposed to drive tumor development in over 70% of human cancers, such as colorectal cancer, breast cancer, prostate cancer, and liver cancer. However, finding disease-specific MYC inhibitors has been challenging; despite extensive efforts, drugs targeting MYC remain unavailable for clinical use. The lack of specific MYC inhibitors also makes it difficult to determine how MYC dysregulation affects disease states beyond cancer. 6，7 .

[0004] Recently discovered Lon protease produced by urinary tract pathogenic Escherichia coli can degrade C-MYC, attenuate the expression of C-MYC-dependent genes, and delay tumor progression. This study investigated the response of MYC to bacterial kidney infection and the protective potential of LON therapy for this indication. Recombinant Lon protease (rLOn) was identified as a potent pan-MYC inhibitor that protects susceptible individuals. Irf3- / - Mice were protected from kidney tissue pathology and bacterial clearance was accelerated. In rLon-treated Apc... Min / + Similar effects were detected in cancerous tissues of mice, where rLOn treatment inhibited the overexpression of C-MYC, N-MYC, and L-MYC. Through peptide mapping, the LON peptidase domain was shown to degrade C-MYC, N-MYC, and L-MYC in human kidney cells, and the N-terminal domain (NTD) was identified as an inhibitor of L-MYC transcription. These results indicate that rLOn and its related peptides are a highly potent pan-MYC inhibitor with a dual mechanism of action and marginal effects on healthy tissues, suggesting significant therapeutic potential. Summary of the Invention

[0005] The present invention provides a peptide comprising an amino acid sequence substantially identical to one or more domains of a Lon protease or a functional fragment or variant thereof.

[0006] Lon proteases are ATP-dependent serine peptidases. Examples of Lon proteases include those secreted by bacteria, such as *Escherichia coli*. The peptides of the present invention may comprise an amino acid sequence substantially identical to the amino acid sequence of one or more domains of bacterial-derived Lon proteases. Our meaning is that these molecules have bacterial-derived structures, thus encompassing synthetic replicas of bacterial-derived molecules. For example, in the case of proteins, the proteins may be biosynthesized by bacteria, recombinantly biosynthesized as exogenous molecules in an artificial expression system, or artificially synthesized proteins with sequences matching bacterial proteins. Inhibitors are available from a variety of bacteria, including, for example, *Streptococcus*, *Pseudomonas*, *Shigella*, *Campylobacter*, and *Salmonella*. In a particular embodiment, the bacteria are urinary tract pathogens, preferably *Escherichia coli*, more preferably *Escherichia coli* 536 or CFT073.

[0007] When the domain is an N-terminal domain, Lon proteases typically originate from prokaryotes, particularly bacteria.

[0008] The table below provides other non-restrictive examples of Lon proteases. Table 1

[0009] The amino acid sequence of the Lon protease from the example Escherichia coli strain is as follows: (SEQ ID No. 1).

[0010] Lon proteases contain an N-terminal domain, an AAA+ domain, and a C-terminal proteolytic domain or peptidase. Those skilled in the art can use the methods described in the Examples section to determine the domains in any Lon protease.

[0011] The N-terminal domain typically includes an α-helical region. In Lon proteases derived from *E. coli*, the N-terminal domain is usually located between amino acid 1 and 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, or 310. The α-helix is ​​typically located between amino acids 100, 105, 110, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 and 230, 235, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305 or 310.

[0012] In Lon proteases derived from E. coli, the AAA+ domain is typically located between amino acids 230, 235, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, or 310 and 560, 565, 570, 575, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 595, 600, 605, or 610.

[0013] In Lon proteases derived from E. coli, the C-terminal proteolytic domain is typically located between amino acids 560, 565, 570, 575, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 595, 600, 605, or 610 and 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, or 790.

[0014] In the peptides of the present invention, the Lon protease domain may be an N-terminal domain, which may include or exclude an α-helix, an AAA+ domain, or a C-terminal domain, in whole or in part.

[0015] Therefore, the present invention provides a peptide comprising an amino acid sequence substantially identical to the amino acid sequence of the N-terminal domain of a Lon protease or a functional fragment or variant thereof. The N-terminal domain may include an α-helical region, or the region may be partially or completely deleted.

[0016] Furthermore, the present invention provides a peptide comprising an amino acid sequence substantially identical to the AAA+ domain of a Lon protease or a functional fragment or variant thereof.

[0017] Furthermore, the present invention provides a peptide comprising an amino acid sequence substantially identical to the amino acid sequence of the C-terminal proteolytic domain of a Lon protease or a functional fragment or variant thereof.

[0018] In one embodiment, the peptide comprises an amino acid sequence substantially identical to the amino acid sequence of a first Lon protease domain or a functional fragment or variant thereof, and an amino acid sequence substantially identical to the amino acid sequence of a second Lon protease domain or a functional fragment or variant thereof. In other words, a portion of the amino acid sequence of the peptide is substantially identical to the amino acid sequence of a first Lon protease domain or a functional fragment or variant thereof, and another portion is substantially identical to the amino acid sequence of a second Lon protease domain or a functional fragment or variant thereof.

[0019] In one embodiment, it comprises an amino acid sequence substantially identical to only one or only two domains of a Lon protease or a functional fragment or variant thereof. The peptide may contain other amino acids, or may consist of an amino acid sequence substantially identical to one or two Lon protease domains.

[0020] Optionally, the peptide may comprise an amino acid sequence substantially identical to the amino acid sequence of the N-terminal domain of a Lon protease or a functional fragment or variant thereof, and an amino acid sequence substantially identical to the amino acid sequence of another domain (such as the C-terminal proteolytic domain of a Lon protease or a functional fragment or variant thereof).

[0021] Optionally, the peptide may comprise an amino acid sequence substantially identical to the amino acid sequence of the C-terminal proteolytic enzyme of Lon protease or a functional fragment or variant thereof, and an amino acid sequence substantially identical to the amino acid sequence of another domain, such as the N-terminal domain of Lon protease or a functional fragment or variant thereof.

[0022] In one embodiment, the peptide comprises an amino acid sequence substantially identical to the amino acid sequence of a first Lon protease domain or a functional fragment or variant thereof, and an amino acid sequence substantially identical to the amino acid sequence of a second Lon protease domain or a functional fragment or variant thereof. In other words, a portion of the amino acid sequence of the peptide is substantially identical to the amino acid sequence of a first Lon protease domain or a functional fragment or variant thereof, and another portion is substantially identical to the amino acid sequence of a second Lon protease domain or a functional fragment or variant thereof.

[0023] In one embodiment, it comprises an amino acid sequence substantially identical to only one or only two domains of a Lon protease. The peptide may contain other regions, or may consist of an amino acid sequence substantially identical to one or two Lon protease domains.

[0024] This peptide is not typically composed of the amino acid sequence of the wild-type Lon protease.

[0025] The length of the peptide can be, for example, less than approximately 780, 770, 760, 750, 740, 730, 720, 710, 700, 690, 680, 670, 660, 650, 640, 630, 620, 610, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 490, 480, 470, 46 0, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130 or 120 amino acids.

[0026] When a peptide contains an amino acid sequence substantially identical to the N-terminal domain of a Lon protease or a functional fragment or variant thereof, its length may optionally exceed 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 236, 238, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 252, or 254 amino acids. Its length can also, optionally, or selectively be less than 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 249, 248, 247, 246, 245, 244, 243, 242, 241, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 129, 128, 127, 126, 125, 124, 123, 122, 121, 120, 119, 118, 117, 116, or 115 amino acids.

[0027] When a peptide contains an amino acid sequence substantially identical to the C-terminal proteolytic domain of a Lon protease or a functional fragment or variant thereof, its length may optionally exceed 180, 185, 190, 195, 200, 205, 210, 215, or 220 amino acids. Its length may also, optionally, or preferably be less than 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, or 120 amino acids.

[0028] When a peptide contains an amino acid sequence that is substantially identical to the amino acid sequence of the N-terminal domain of a Lon protease or a functional fragment or variant thereof, and an amino acid sequence that is substantially identical to the amino acid sequence of the peptidase domain of a Lon enzyme or a functional fragment or variant thereof, these sequences may be continuous or separated by other amino acids.

[0029] In one implementation, the N-terminal domain has one of the following sequences: MNPERSERIEIPVLPLRDVVVYPHMVIPLFVGREKSIRCLEAAMDHDKKIMLVAQKEASTDEPGVNDLFTVGTVASILQMLKLPDGTVKVLVEGLQRARISALSDNGEHFSAKAEYLE (SEQ ID No. 2 (excluding α-helix)), and MNPERSERIEIPVLPLRDVVVYPHMVIPLFVGREKSIRCLEAAMDHDKKIMLVAQKEASTDEPGVNDLFTVGTVASILQMLKLPDGTVKVLVEGLQRARISALSDNGEHFSAKAEYLESPTIDE REQEVLVRTAISQFEGYIKLNKKIPPEVLTSLNSIDDPARLADTIAAHMPLKLADKQSVLEMSDVNERLEYLMAMMESEIDLLQVEKRIRNRVKKQMEKSQREYYLNEQMKAIQKELGEMD (SEQ ID No. 3 (containing alpha helix)).

[0030] Therefore, the present invention provides a peptide comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID No. 2 or 3, or a fragment or variant thereof. Identification, fragments, and variants are as defined with respect to other embodiments or aspects of the invention.

[0031] In one implementation, the AAA+ domain comprises the following amino acid sequence: DAPDENEALKRKIDAAKMPKEAKEKAEAELQKLKMMSPMSAEATVVRGYIDWMVQVPWNARSKVKKDLRQAQEILDTDHYGLERVKDRILEYLAVQSRVNKIKGPILCLVGPPGVGKTSLGQSIAKATGRKYVRMALGGVRDEAEIRGHRRTYIGSMPGKLIQKMAKVGVKNPLFLLDE IDKMSSDMRGDPASALLEVLDPEQNVAFSDHYLEVDYDLSDVMFVATSNSMNIPAPLLDRMEVIRLSGYTEDEKLNIAKRHLLPKQIERNALKKGELTVDDSAIIGIIRYYTREAGVRGLEREISKLCRKAVKQLLLDKSLKHIEINGDNLHDYLGVQRFDYGRADNENRVGQVTG (SEQ ID No. 4).

[0032] Therefore, the present invention provides a peptide comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID No. 4 or a fragment or variant thereof.

[0033] In one implementation, the C-terminal domain comprises the following amino acid sequence: LAWTEVGGDLLTIETACVPGKGKLTYTGSLGEVMQESIQAALTVVRARAEKLGINPDFYEKRDIHVHVPEGATPKDGPSAGIAMCTALVSCLTGNPVRADVAMTGEITLRGQVLPIGGLKEKLLAAHRGGIKTVLIPFENKRDLEEIPDNVIADLDIHPVKRIEEVLTLALQNEPSGMQVVTAK (SEQ ID No. 5).

[0034] Therefore, the present invention provides a peptide comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID No. 5 or a fragment or variant thereof.

[0035] Optionally, the peptide may contain an amino acid sequence substantially identical to the amino acid sequence of SEQ ID.2 or 3 and 5, or a fragment or variant thereof.

[0036] The term "fundamental consistency" means that a sequence is similar to a reference sequence. For example, fundamental consistency may mean approximately or at least about 60%, 65%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity or homology.

[0037] Therefore, the peptide may contain an amino acid sequence that is about or at least about 60%, 65%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or homologous to the amino acid sequence of the N-terminal domain of the Lon protease (such as SEQ ID No. 2 or 3) or a functional fragment or variant thereof. The N-terminal domain may contain an α-helix, or they may be deleted in whole or in part.

[0038] Alternatively, the peptide may comprise an amino acid sequence that has about or at least about 60%, 65%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or homology with the amino acid sequence of the C-terminal proteolytic domain (e.g., with SEQ ID No. 5 of Lon protease or a functional fragment or variant thereof).

[0039] Alternatively, the peptide may contain an amino acid sequence (such as SEQ ID No. 4) of the AAA+ domain of the Lon protease or a functional fragment or variant thereof having about or at least about 60%, 65%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or similarity.

[0040] Alternatively, the peptide may comprise an amino acid sequence that is about or at least about 60%, 65%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the C-terminal proteolytic domain (such as SEQ ID No. 5 of the Lon protease), and an amino acid sequence that is about or at least about 60%, 65%, 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the C-terminal proteolytic domain (such as SEQ ID No. 5 of the Lon protease).

[0041] The variant preferably has at least about 40% homology with the amino acid sequence of the peptide over its entire length. More preferably, the variant may have at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% homology over its entire sequence.

[0042] The variant preferably has at least about 40% homology with the amino acid sequence of the peptide over its entire length. More preferably, the variant may have at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% homology over its entire sequence.

[0043] Over a stretch of 75 or more consecutive amino acids of a peptide, such as 80, 90, 100, 125, 150, 175 or 200 or more, there may be at least about 80%, such as at least about 85%, about 90%, about 95%, about 98% or about 99% homology or similarity (“hard homology”).

[0044] Standard methods in this field can be used to determine homology or similarity. For example, the UWGCG software package provides the BESTFIT program, which can be used to calculate homology, for example, using its default settings (Devereux et al. (1984) NucleicAcids Research 12, pp. 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or sequence alignment (e.g., to identify equivalent residues or corresponding sequences (usually at default settings)), as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al. (1990) Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0045] This variant may contain one or more amino acid substitutions, for example, at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more substitutions. The one or more substitutions are preferably one or more conservative substitutions. Conservative substitutions replace the amino acid with other amino acids that have similar chemical structures, similar chemical properties, or similar side chain sizes. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid variations are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table 2 below. If the amino acids have similar polarity, the hydrophilicity scale of the amino acid side chains in Table 3 can also be used to determine this.

[0046] Table 2: Chemical properties of amino acids

[0047] Table 3 - Hydrophilicity Scale

[0048] Methods for introducing or substituting natural amino acids are well known in the art. For example, methionine (M) is replaced with arginine (R) by substituting the methionine codon (ATG) with the arginine codon (CGT) at the relevant position of the polynucleotide encoding the variant. Methods for introducing or substituting non-natural amino acids are also well known in the art. For example, non-natural amino acids can be introduced by including synthetic aminoacyl-tRNA in an IVTT system used to express mutant monomers. Alternatively, they can be introduced by expressing mutant monomers in E. coli with synthetic (i.e., non-naturally present) analogs of those specific amino acids in the presence of specific amino acid auxotrophs. If mutant monomers are generated through partial peptide synthesis, they can also be generated by naked ligation.

[0049] One or more amino acids may be deleted from the peptides described above. A maximum of 1, 2, 3, 4, 5, 10, 20, or 30 or more amino acids may be deleted.

[0050] The length of the fragment can be at least 50, at least 100, at least 150, at least 200, or at least 250 amino acids.

[0051] One or more amino acids may be added alternatively or additionally to the peptides described above. An extension may be provided at the amino or carboxyl terminus of the peptide, variant, or fragment. The extension may be short, for example, 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example, up to 50 or 100 amino acids. A carrier peptide or protein may be fused to the peptides of the present invention.

[0052] Fragments or variants generally have a function. A functional fragment or variant refers to a fragment or variant that exhibits the same function as the peptide of the present invention. For example, a functional fragment of the N-terminal domain of a Lon protease inhibits L-MYC, C-MYC, and N-MYC. A functional fragment of the C-terminal proteolytic domain of a Lon protease degrades C-MYC, N-MYC, and L-MYC. Methods for measuring the activity of wild-type peptides are known in the art and are provided in the examples. The activity of variants may be increased or decreased compared to wild-type peptides, for example, by about 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more.

[0053] Peptides, variants, or fragments can be isolated, substantially isolated, purified, or substantially purified. If a peptide, variant, or fragment contains no other components, such as lipids or other peptides, it is isolated or purified. If a peptide, variant, or fragment is mixed with a carrier or diluent that does not interfere with its intended use, it is substantially isolated or substantially purified. For example, if a peptide, variant, or fragment exists in a form containing less than 10%, less than 5%, less than 2%, or less than 1% of other components, it is substantially isolated or substantially purified.

[0054] Throughout the discussion in this paper, the standard single-letter codons for amino acids were used. These are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). The standard substitution notation, DXXN, indicates that the D at position XX is replaced by N.

[0055] Any peptides described herein, including variants and fragments, can be modified to aid in their identification or purification, for example, by adding histidine residues (his tags), aspartic acid residues (asp tags), streptavidin tags, flag tags, SUMO tags, GST tags, or MBP tags, or by adding signaling sequences to promote their secretion from cells in which the peptide is naturally absent. Another method of introducing genetic tags is to chemically react the tag to a natural or engineered site on the peptide. One example is reacting a gel transfer reagent with an externally engineered cysteine ​​residue on the peptide.

[0056] Any peptide described herein, including variants and fragments, can be labeled with a display label. A display label can be any suitable label that allows for the detection of the peptide. Suitable labels include, but are not limited to, fluorescent molecules, radioactive isotopes (e.g., 125I, 35S), enzymes, antibodies, antigens, polynucleotides, and ligands (e.g., biotin).

[0057] Any peptide described herein, including variants and fragments, can be prepared synthetically or recombinantly. For example, peptides can be synthesized via in vitro translation and transcription (IVTT). The amino acid sequence of a peptide can be modified to include non-naturally occurring amino acids or to increase the peptide's stability. Such amino acids can be introduced during the production process when peptides are produced synthetically. The polynucleotide sequence encoding the peptide can be derived and replicated using standard methods in the art. The polynucleotide sequence encoding the peptide can be expressed in bacterial host cells using standard techniques in the art. This will be discussed in more detail below. Peptides may also be altered after synthetic or recombinant production.

[0058] Peptides can also be produced using D-amino acids. For example, a peptide may comprise a mixture of L-amino acids and D-amino acids. This is a conventional method for producing such peptides in the art.

[0059] Peptides may also contain other nonspecific modifications, as long as they do not interfere with the peptide's function. Many nonspecific side-chain modifications are known in the art, and the side chains of peptides can be modified. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with a methylacetylimine ester, or acylation with acetic anhydride.

[0060] Peptides can be mass-produced after purification from protein-producing organisms or recombinant expression using any protein liquid chromatography system. Typical protein liquid chromatography systems include FPLC, AKTA system, Bio-Cad system, Bio-Rad BioLogic system, and Gilson HPLC system.

[0061] The present invention further provides a polynucleotide encoding the peptide of the present invention.

[0062] The term polynucleotide refers to a polymer containing two or more nucleotides. Preferably, the polynucleotide contains at least 30, 40, 50, or 100 nucleotides. Nucleotides can be naturally occurring or artificially synthesized.

[0063] Nucleotides typically consist of a nucleobase, a sugar, and at least one linker group, such as a phosphate group, a 2'O-methyl group, a 2'methoxyethyl group, an aminophosphate group, a methylphosphonate group, or a thiophosphate group. The nucleobase is usually a heterocyclic ring. Nucleosides include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is usually a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymine, deoxyuridine, and deoxycytidine. Nucleosides may be adenosine, guanosine, uridine, and cytidine.

[0064] Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides can be deoxyribonucleotides. Nucleotides usually contain monophosphate, diphosphate, or triphosphate. The phosphate can be attached to the 5' or 3' side of the nucleotide.

[0065] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), and deoxyadenosine diphosphate (dA). DP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotide can be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP, or dCMP. In some embodiments, the nucleotide is selected from dAMP, dTMP, dGMP, or dCMP.

[0066] Nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'-aminopyrimidines (such as 2'-aminocytidine and 2'-aminouridine), 2'-hydroxypurines (such as 2'-fluoropyrimidines (such as 2'-fluorocytidine and 2'-fluorouridine)), hydroxypyrimidines (such as 5'-α-P-boraneuridine), 2'-O-methylnucleotides (such as 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine and 2'-methyluridine), and 4'-thiopyrimidines (such as 4'-thiouridine and 4'-thiocytidine), as well as nucleotides with nucleobase modifications (such as 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)uridine, and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0067] One or more nucleotides in a polynucleotide can be modified, for example, by using a label or tag. The label can be any suitable marker that allows the detection of the nucleotide. Suitable labels include, but are not limited to, fluorescent molecules, radioactive isotopes (e.g., fluorescent molecules, radioactive isotopes, etc.). 125 I, 35 S), enzymes, antibodies, antigens, other polynucleotides and ligands (such as biotin).

[0068] Nucleotides in polynucleotides can be linked to each other in any way. Nucleotides can be linked by phosphate, 2'O-methyl, 2'-methoxyethyl, aminophosphate, methylphosphonate, or thiophosphate. Nucleotides are usually linked by their sugar and phosphate groups. Nucleotides can also be linked by their nucleobases, as in pyrimidine dimers.

[0069] Polynucleotides may include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Preferably, polynucleotides include DNA. Polynucleotides may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), morpholinonucleotide, or other synthetic polymers having nucleotide side chains.

[0070] Alternatives to practices that can be used for codon optimization and codon wobble, both of which are known to those skilled in the art, are also conceivable. Therefore, it is understood that both codon-optimized and codon-wobble-based isolated polynucleotides can also be envisioned. In some embodiments, the polynucleotide is codon-optimized for human expression.

[0071] Polynucleotides can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences. Direct chemical synthesis of polynucleotides can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109; the diethylphosphite method of Beaucage et al., 1981, Tetra. Lett., 22:1859; and the solid-support method of U.S. Patent No. 4,458,066. Introducing mutations into polynucleotide sequences via PCR can be performed as described, for example, in the following literature: PCR Technology: Principles and Applications for DNA Amplification, HAErlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1:17.

[0072] The present invention also provides a vector comprising the polynucleotide of the present invention.

[0073] The present invention also provides a host cell comprising the polynucleotide of the present invention or the vector of the present invention.

[0074] Various viral and nonviral vectors are known to those skilled in the art. Nonviral vectors include plasmids, episomal vectors, and human artificial chromosomes (see, for example, Harrington et al., 1997, Nat Genet. 15:345). For example, nonviral vectors that can be used to express the amiRNA and CAR of the present invention in mammalian (e.g., human) cells include pThioHis A, B, and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invitrogen, San Diego, CA), MPSV vectors, and many other vectors known in the art for expressing other peptides or proteins. Useful viral vectors include retrovirus-based, adenovirus-based, adeno-associated virus-based, herpesvirus-based, SV40-based vectors, papillomavirus-based, HBP EpsteinBarr virus, vaccinia virus vectors, and Semliki Forest Virus (SFV). See above Brent et al.; supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143. In particular, retroviral, lentiviral, adenoviral, or adeno-associated virus vectors are commonly used for expression in immune cells such as T cells. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737).

[0075] In some embodiments, the vector is a retroviral or lentiviral vector. Optionally, the vector is an SFG retroviral vector. In some embodiments, the vector is a lentiviral vector. Lentiviral vectors include self-inactivating lentiviral vectors (so-called SIN vectors).

[0076] The choice of expression vector depends on the intended host cell in which the vector will be expressed. Expression vectors for mammalian host cells may include expression control sequences such as origin of replication, promoters, and enhancers (see, for example, Queen et al., 1986, Immunol. Rev. 89:49-68), as well as necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or modulotropic or tunable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPS V promoters, tetracycline-inducible CMV promoters (such as human immediate early CMV initiator), constitutive CMV initiators, EF1α promoters, phosphoglycerate kinase (PGK) promoters, and promoter-enhancer combinations known in the art.

[0077] Cultures of transformed organisms can be expanded under non-inducible conditions without biasing the population towards sequences encoding their expressed products that are better tolerated by host cells. In addition to promoters, other regulatory elements may also be necessary or desirable for the efficient expression of the peptides of the present invention. These elements typically include the ATG start codon and adjacent ribosome binding sites or other sequences. Furthermore, expression efficiency can be improved by adding enhancers suitable for the cell system used (see, for example, Scharf et al., 1994, Results Probl. Cell Differ. 20:125; and Bittner et al., 1987, Meth. Enzymol. 153:516). For example, the SV40 enhancer or CMV enhancer can be used to increase expression in mammalian host cells.

[0078] In some implementations, the non-coding region of the vector contains multiple nucleotides. For example, the non-coding region of a retroviral vector may contain multiple nucleotides.

[0079] Genetic engineering of host cells can be performed using standard cloning and expression techniques known in the art (e.g., as described in Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The vectors of the present invention can be introduced into host cells using such techniques.

[0080] The various forms of the term "transfection" are intended to encompass a range of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE dextran transfection, etc.

[0081] Representative host cells include many Escherichia coli strains, mammalian cell lines such as CHO, CHO-K1 and HEK293; insect cells such as Sf9 cells; and yeast cells such as Saccharomyces cerevisiae and Pichia pastoris.

[0082] In some embodiments, the host cell is a mammalian host cell. For example, the host cell may be a mouse, rat, human, dog, cat, horse, cattle, primate, goat, or sheep host cell. In some embodiments, the host cell is a human or mouse host cell, preferably a human host cell.

[0083] The cell lines that can be used include the NK cell line NK-92.

[0084] The mammalian host cells covered by this invention include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with DH FR selective markers, such as those described in RJ Kaufman and PA Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In some embodiments, the host cell is CHO K1PD cells. In some embodiments, the host cell is NSO1 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system shown in WO 87 / 04462, WO 89 / 01036, and EP 338841.

[0085] In some embodiments, the host cell is an immune response cell. In the context of this invention, an immune response cell is an immune cell. Typically, immune response cells can participate in an immune response, preferably an inflammatory immune response, such as a response to cancer.

[0086] A pharmaceutical composition comprising the peptide or polynucleotide or carrier of the present invention, and a pharmaceutically acceptable carrier, is also provided.

[0087] Carriers and / or diluents are typically selected to suit the intended route of administration and may include agents for altering, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. Typically, these carriers and / or diluents comprise aqueous solutions or alcohol / aqueous solutions, emulsions, or suspensions, including saline and / or buffer media.

[0088] Other suitable agents included in the pharmaceutical composition include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antibacterial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), fillers (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as free serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, and hydrophilic polymers (such as poly… Vinylpyrrolidone), low molecular weight peptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as Pluronic; PEG; sorbitol esters; polysorbates, such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancers (such as sucrose or sorbitol), tension enhancers (such as alkali metal halides, such as sodium chloride or potassium chloride or mannitol sorbitol), delivery carriers, excipients, and / or drug adjuvants.

[0089] The carrier and / or diluent may be a parenteral or, optionally, an intravenous carrier. Suitable parenteral carriers include sodium chloride solution, Ringer's glucose, glucose and sodium chloride, and lactated Ringer's solution. Suitable physiologically acceptable thickeners such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin, and alginate may be included. Intravenous carriers include fluid and nutritional supplements and electrolyte supplements, such as Ringer's glucose-based supplements. In some cases, agents that adjust the tension of the composition may be included, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride in the pharmaceutical composition. For example, in many cases, it is ideal for the composition to be substantially isotonic. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. The exact formulation will depend on the route of administration. Other relevant principles, methods, and components of pharmaceutical formulations are well known (see Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd edition).

[0090] The pharmaceutical compositions of the present invention can be administered via one or more routes of administration using one or more of the various methods known in the art. Those skilled in the art will understand that the route and / or mode of administration will vary depending on the desired outcome. Routes of administration for the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoral, spinal, or other parenteral routes of administration, such as by injection or infusion. The phrase “parenteral administration” as used herein refers to a method of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrasheathal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous corneal, intra-articular, subcapsular, subarachnoid, spinal, epidural, intratumoral, intrapleural, and intrasternal injections and infusions. In some embodiments, the pharmaceutical composition is administered intratumorally. In other embodiments, administration is intrapleural or intraperitoneal. When considering parenteral administration, the pharmaceutical composition is typically in a sterile, pyrogen-free, parenterically acceptable compositional form. A particularly suitable carrier for parenteral injection is a properly preserved sterile isotonic solution. The pharmaceutical composition may be in the form of a lyophilized product, such as a lyophilized cake.

[0091] Alternatively, the pharmaceutical compositions of the present invention can be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration.

[0092] In some embodiments, the pharmaceutical composition is intended for subcutaneous administration. Typically, pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids such as methionine, and / or sugars such as sucrose), buffers, and tonic modulators. Alternatively, the pharmaceutical composition may be intended for intravenous administration.

[0093] Further, peptide, polynucleotide, carrier, or pharmaceutical compositions comprising the peptides of the present invention are provided for therapeutic purposes. In particular, the peptides, polynucleotides, carriers, or compositions can be used to treat infections, especially bacterial infections, or to treat cancer.

[0094] Cancer can be any cancer, such as, but not limited to, cancers found in the gastrointestinal tract, including colorectal cancer, oral cancer, brain cancer, lung cancer, kidney cancer, vaginal cancer, bladder cancer, liver cancer, skin cancer, breast cancer, prostate cancer, and / or ovarian cancer.

[0095] Infections can be any kind of infection, especially bacterial infections, such as those of the bladder, kidneys, or urinary tract.

[0096] A method for treating infection or cancer is also provided, comprising the step of administering an effective amount of the peptide, polynucleotide, carrier, or pharmaceutical composition of the present invention to a subject in need.

[0097] Further, the present invention provides peptides or pharmaceutical compositions comprising the present invention for treating diseases regulated by L-MYC, N-MYC, or C-MYC, or for treating diseases regulated by L-MYC, N-MYC, and C-MYC.

[0098] A method for treating a disease regulated by L-MYC, N-MYC, or C-MYC is also provided, comprising the step of administering an effective amount of the peptide or pharmaceutical composition of the present invention to a subject in need.

[0099] The present invention also provides a peptide comprising an amino acid sequence substantially homologous to an amino acid sequence of a Lon protease or a functional variant or fragment thereof, for the treatment of diseases such as infections or cancer, wherein the diseases are regulated by L-MYC or N-MYC.

[0100] In the description and claims of this specification, the words “comprising” and “including,” and variations thereof, such as “comprising” or “including,” mean “including, but not limited to,” and do not exclude other components, integers, or steps. Furthermore, unless the context requires otherwise, the singular includes the plural: particularly in the case of the use of the indefinite article, unless the context requires otherwise, the specification should be understood to consider both the plural and the singular.

[0101] In all cases, the terms "include" or "contain" are included and can be replaced by "consistsof" or "consisting of".

[0102] Preferred or optional features of each aspect of the invention may be as described in any other related aspect. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives listed in the foregoing paragraphs, claims, and / or the following description and drawings, particularly their respective features, may be employed independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination, unless these features are incompatible.

[0103] When referring to measurable values ​​(such as quantities, percentages, etc.), the term "about" as used herein means a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, from a specified value, as these variations are suitable for performing the disclosed methods. Where the term "about" appears in all instances involving measurable values, this disclosure includes examples or embodiments where the word "about" is not used. Attached Figure Description

[0104] One or more embodiments of the invention will now be described by way of example only with reference to the accompanying drawings, wherein:

[0105] Figure 1 The inhibitory effect of rLon protease treatment on c-MYC.

[0106] a, for E. coli CFT073 infection Irf3- / - , Irf7- / -Schematic diagram of the rLon protease treatment regimen in C57BL / 6WT mice. Mice were treated with rLon (intraperitoneal injection (100 μL, 250 μg / mL), once daily for seven days, starting one day before E. coli CFT073 bladder infection). b, Infection-regulated MYC-dependent genes in the kidneys of placebo mice. 497 genes were activated, and 256 genes were repressed. c, Volcano plot showing strong activation of the total number of regulated genes in the kidneys of infected mice (red, upregulated; blue, downregulated; gray, not significantly regulated; critical FC ≥ 2, P < 0.05 relative to uninfected mice). d, Inhibition of the infection response by rLon treatment. In rLon-treated mice, the number of MYC-dependent genes (2 genes activated, compared to 753 genes in placebo) and the total number of regulated genes (43 regulated genes, compared to 3696 genes in placebo) were significantly reduced. e. c-MYC protein staining in kidney tissue sections from rLon-treated mice compared to the placebo group. Confocal microscopy; green, MYC; blue, nucleus; scale bar, 10 μm. f. Quantification of c-MYC protein staining in e (n = 3 uninfected mice, n = 7 placebo 24 h, n = 5 rLon 24 h, n = 3 placebo 7 d, n = 4 rLon 7 d). The straight line represents the median, corrected for Dunn after one-way ANOVA. g. Suppression of the innate immune response to infection by rLon treatment. Z-scores of activation for each pathway are shown. h. Activation of the MYC-dependent innate immune response in the placebo group. i. Suppression of the MYC-dependent innate immune response pathway and inflammation in rLon-treated mice compared to the placebo group. Orange, upregulated; blue, downregulated.

[0107] Figure 2 The therapeutic effects of rLon in a mouse model of acute pyelonephritis.

[0108] a, b, compared to the placebo group Irf3- / - Treatment of mice with rLon protease (see Figure 1 (Illustrative diagram in the image) reduced gross renal pathology (arrows indicate abscess, scale bar, 5mm, left kidney). c, improved rLon treatment Irf3- / - d. Bacterial clearance rate in mouse (right kidney) renal tissue. e. Decreased bacterial count in urine. f. Inhibition of inflammation, quantified as the number of neutrophils in urine (red, placebo; blue, rLon). g. Results in the placebo group, showing infection. Irf3- / - The disease phenotype in mice is characterized by extensive neutrophil infiltration and microabscess formation in infected kidneys. Whole kidney sections were stained with hematoxylin and eosin (H&E). g, compared to healthy kidneys, the infected kidneys... Irf3- / -The significant increase in kidney size in mice was reversed in rLon-treated mice. h, Histological sections from rLon-treated Irf3- / - mice showed reduced kidney size, less pathological changes, and decreased neutrophil infiltration compared to the placebo group. i, Co-localization of bacteria and neutrophils in pathological areas in the placebo group (n=4 mice per group; red, neutrophils; green, bacteria; blue, nuclei; scale bar, see figure). j, Reduced bacterial and neutrophil staining observed by immunohistochemistry in rLon-treated mice (n=4 mice per group; red, neutrophils; green, bacteria; blue, nuclei; scale bar, see figure). k, quantitative data of neutrophils in kidney tissue sections and i, j, quantitative data of bacterial staining in kidney tissue sections are expressed as mean ± sd(b, c, k) or sem(d, e), analyzed by Kruskal-Wallis with Dunn correction (b, c, k), or by two-way ANOVA followed by Bonferr-Loni's multiple comparison test (d, e). P < 0.001.

[0109] Figure 3 rLon Irf3- / - The study showed an inhibitory effect on nephrotoxicity in mice, but no toxicity in uninfected mice.

[0110] a. Escherichia coli CFT073 infection Irf3- / - Gene expression associated with nephrotoxicity, kidney injury, or kidney-specific cell death in mice. b, Compared to uninfected mice, MYC and IRF7-dependent genes regulating kidney injury or kidney-specific cell death were strongly upregulated in the placebo group. b, Compared to the placebo group, rLon-treated mice completely suppressed genes regulating kidney injury or kidney-specific cell death. c, Inhibition of kidney injury, damage, and cell death pathways by rLon treatment. d, Top-ranking regulatory genes for nephrotoxicity. (Except for...) Lyz Furthermore, genes regulated in the placebo group were not regulated in the rLon-treated group. e. Principal component analysis of whole kidney mRNA profiles. The right cluster shows uninfected mice (green) and adjacent rLon-treated mice (purple). The left cluster shows the placebo group (red). F. Heatmap of total detected genes and all nephrotoxic genes, comparing uninfected control group with placebo and rLon mice (fold change compared to uninfected group: white, no change; red, activation; blue, inhibition). The transcriptomic profile of the rLon-treated group was similar to that of the uninfected control group, but lighter in color. Compared to uninfected mice, placebo mice showed strong gene regulation.

[0111] Figure 4 L-Myc and N-Myc are involved in disease responses to infection and cancer.

[0112] ab, Network analysis of L-Myc-dependent genes regulated by *E. coli* CFT073 infection. rLon treatment suppressed the L-Myc response, with only one differentially expressed L-Myc-dependent gene at 24 hours and no differential expression at 7 days. c, Immunohistochemical visualization of reduced L-Myc staining in kidney sections obtained from rLon-treated mice (n=4 mice per group; green, L-Myc; blue, cell nuclei; scale bar, see figure). n=2 mice per group, critical FC ≥ 2, P < 0.05 relative to uninfected mice.

[0113] de. Network analysis of N-Myc-dependent genes regulated by *E. coli* CFT073 infection. rLon treatment suppressed the N-Myc response, with only 10 N-Myc-dependent genes showing differential expression at 24 hours and 2 at 7 days. f. Reduced N-Myc staining in kidney sections obtained from rLon-treated mice, visualized by immunohistochemistry (n=4 mice per group; green, L-Myc; blue, cell nuclei; scale bar, see figure). n=2 mice per group, critical FC ≥ 2, P < 0.05 relative to uninfected mice. g. Treatment of colon cancer-prone mice with rLon protease. Apc Min / + A schematic diagram of the mouse protocol. Mice were administered rLon (100 μL, 250 μg / mL) orally twice daily for 14 days, starting at 11 weeks of age. Compared to the disease-free C57BL / 6 control group, Apc Min / + Strong activation of c-Myc, N-Myc, and L-Myc-dependent genes in mice. km,rLon treatment Apc Min / + Regulation and repression of c-Myc, N-Myc, and L-Myc-dependent genes were reduced in mice. The critical FC was ≥ 2 (P < 0.05) relative to healthy C57BL / 6WT mice, with n=2 mice per group.

[0114] Figure 5 LON-MYC molecular data

[0115] a. Structural overview of the Lon protease, highlighting its functional domains. b. Western blot of whole-cell extracts from human renal cancer cells exposed to recombinantly expressed full-length Lon, N-terminal domain (NTD), ATPase domain (AAA), or peptidase domain (Pep). Anti-Lon antibody. c. Cellular uptake of full-length Lon, NTD, AAA, or peptidase domains labeled with AlexaFluor by human lung cancer cells. Cytoplasmic uptake and perinuclear accumulation. Magenta, labeled protein; blue, nucleus. df. Analysis of MYC levels in cells treated with full-length Lon, N-terminal domain (NTD), ATPase domain (AAA), or peptidase domain (Pep). d. Reduced c-Myc levels in total cell and nuclear extracts. e. Reduced N-Myc levels in total cell and nuclear extracts. f. Reduced L-Myc levels in total cell and nuclear extracts. g, PCR quantification of L-Myc, N-Myc, and c-Myc mRNA in cells exposed to full-length Lon, NTD, AAA, or peptidase domain peptides. L-Myc ( MYCL-VA Significant inhibition. Fold change compared to PBS control cells. Hi, computer docking of NTD peptides with c-Myc and N-Myc was performed using Alfafold. Strong binding of NTD to the leucine-rich zipper domain of MYC was predicted (IPTM=0.697).

[0116] Figure 6 Cellular interactions of rLon and rLon domains and their effects on c-MYC, L-MYC and N-MYC expression

[0117] a, b, AlphaFold multimers predict the interaction between Lon-NTD and the C-terminal portion of C-MYC (residues 356-439) and N-MYC (residues 354-464). c, Biacore analysis shows the dose-dependent interactions of full-length Lon and NTD peptides with His-tagged c-Myc and N-Myc captured on the sensor chip NTA, and 1:1 Langmuir fitting curves. Each sensing plot shows the fitting values ​​(K... D d. The overlap of NTD and MAX indicates that NTD targets the same binding sites in MYC and MAX. e. The inhibition of c-MYC activation by NTD and AAA+ was quantified in an ELISA-based assay by the binding of the MYC / MAX complex to DNA. f, g. Strong inhibition of the universal transcriptional pathway by NTD and AAA+ in human renal cancer cells treated with NTD and AAA+ domain peptides (fold change FC ≥ 1.5, n = 2 experiments).

[0118] Figure 7Structural-functional analysis of the interaction between Lon protein and MYC. a) Structural overview of Lon, highlighting the N-terminal domain, central AAA+ domain, and C-terminal peptidase domain. b) Purity of recombinant Lon, N-terminal domain (NTD), AAA+, and peptidase domain, validated by Western blotting using an anti-Lon antibody. ce) Interaction between Lon-NTD (blue) and the C-terminal portions of (c) c-MYC (residues 356-439, green) and (d) N-MYC (residues 354-464, green). AlphaFold multimers did not predict the interaction between Lon-NTD and (e) L-MYC (lemon green). f) SPR binding analysis showing dose-dependent interactions between full-length Lon and NTD peptides and His-labeled c-Myc and N-myc captured on the sensor chip NTA (magenta) and 1:1 Langmuir fitted curves (black). The table below shows the dissociation constants (K0) of the binding. D g. The overlap between NTD and MAX indicates that NTD targets the same binding sites in MYC and MAX. h. The inhibition of c-MYC activation by NTD and AAA+ was quantified by ELISA-based assays of MYC / MAX complex binding to DNA.

[0119] AlphaFold predicted a low iPTM score of 0.171 for the interaction between full-length c-MYC and full-length Lon protein. AlphaFold predicted the interactions of full-length c-MYC, N-MYC, and L-MYC with the Lon peptidase domain (amino acids 585-784). The putative complexes of all three MYC proteins with the peptidase domain resulted in relatively low iPTM scores (0.324 for c-MYC, 0.32 for N-MYC, and 0.3 for L-MYC).

[0120] Figure 8 Cellular interactions of rLon and rLon domains and their effects on c-MYC, L-MYC and N-MYC expression

[0121] a. Live-cell confocal images show efficient uptake of AZ647-labeled (magenta) rLon (0.902 μM) and its rLon domain (6.314 μM) into the cytoplasm, perinuclear region, and nucleus of A498 cells. Nuclei were counterstained with Hoechst (blue) (quantification based on fluorescence intensity shown in each image; 50 cells per sample; n=2 experiments; scale bar, 3 μm). b. Western blot confirmed the uptake of rLon and its domain in whole-cell extracts of A498 human renal cell carcinoma cells exposed to rLon and its domain peptides. Live-cell confocal images show inhibition of c-MYC and N-MYC protein levels in Lon and NTD-treated cells compared to the control group. L-MYC protein levels were also reduced in peptidase-treated cells compared to the control group. (Green, c-MYC, N-MYC, or L-MYC; Blue, nucleus; scale bar, 10 μm). The following figure shows the quantitative fluorescence intensity (50 cells per sample; n = 2 experiments). Data are expressed as mean ± SD. f, NTD-treated A498 cells. MYCL Strong inhibition of mRNA expression. MYCN Expression was weakly inhibited by the full-length Lon protein and NTD peptide. MYC expression was unaffected. Irf7 Used as a negative control. Data are expressed as mean ± SD. MYC , MYCL and MYCN n = 2-3 experiments; IRF (n=1 experiments). g, h, in human renal cancer cells treated with NTD and AAA+ domain peptides, NTD and AAA+ strongly inhibited the universal transcriptional pathway (fold change FC≥1.5; n=2 experiments).

[0122] Figure 9 Degradation of MYC by Lon and Lon peptide domains. Analysis of protein degradation by ac, rLon (a), peptidase, and AAA+ domains (b). After 120 min, c-MYC (50 ng) was degraded by rLon (0.5 or 1 μg) and by rLon peptidase domain in the presence of AAA+ domain peptides. c-MYC protein controls were shown with and without incubation. This effect was reversed in the presence of protease inhibitors (c). d, e, inhibition of c-MYC, N-MYC, and L-MYC protein expression in whole-cell and nuclear extracts of A498 cells by rLon (d) and NTD peptide (e). f, quantification of the percentage inhibition by Western blot compared to untreated cells. Data are presented as mean. (n = 1–3 experiments).

[0123] Figure 10Rapid response of c-MYC to kidney infection and inhibition of rLon protease therapy.

[0124] a, used for rLon treatment of E. coli CFT073 infection. Irf3- / - Schematic diagram of the mouse protocol. Mice were intraperitoneally injected with rLon (100 μL, 250 μg / ml) once daily for eight days, starting the day before infection with *E. coli* CFT073 in the bladder. Total renal RNA was isolated from infected and uninfected mice at 24 hours and 7 days post-infection, and whole-genome transcriptome analysis was performed to identify disease-related genes by comparing infected mice with uninfected controls. b, c, Response of the placebo group to kidney infection, showing activation of MYC-dependent genes at 24 hours and 7 days post-infection (n = 2 per group; red, upregulated; blue, downregulated; fold change FC ≥ 2, P < 0.05 compared to uninfected mice). d, Inhibition of MYC-dependent response to infection by rLon treatment; significantly reduced number of MYC-dependent genes in rLon-treated mice compared to the placebo group. e, c-Myc protein staining in kidney tissue sections of the rLon-treated group compared to the placebo group. Confocal microscopy; representative images; green, c-Myc; blue, nucleus; scale bar, 20 μm; n = 3–7 mice per group. f, Quantification of c-Myc protein staining from e. Lines represent medians. One-way ANOVA followed by Dunn correction. g, h, Functional analysis of MYC-dependent genes to determine that the innate immune response was activated in the placebo group and suppressed in the rLon treatment group. Orange, upregulated; blue, downregulated. i, Suppression of the innate immune response to infection by rLon treatment. Z-scores of activation for each pathway are shown. Data were normalized against healthy, uninfected mice.

[0125] Figure 11 rLon treatment in infection and cancer models inhibited the activation of L-Myc and N-Myc. a, b, Irf3- / - Network analysis of L-Myc-dependent genes regulated by *E. coli* CFT073 infection in mice. rLon treatment suppressed the L-Myc response to kidney infection, with only one differentially expressed L-Myc-dependent gene after 24 hours and no differential expression after 7 days (n=2 mice per group; critical FC ≥ 2; P < 0.05 compared to uninfected mice). c, d, and rLon treatments suppressed the N-Myc response to kidney infection, with only 10 differentially expressed N-Myc-dependent genes after 24 hours and only 2 differentially expressed after 7 days (n=2 mice per group; fold change FC ≥ 2).

[0126] P < 0.05 compared to uninfected mice). e, f, Immunohistochemical visualization of reduced L-Myc and N-Myc staining in kidney sections obtained from rLon-treated mice (representative images; n = 2–5 mice per group; green, N-Myc or L-Myc; blue, cell nuclei). g, Schematic diagram of the protocol for rLon treatment of colon cancer-susceptible ApcMin / + mice by oral gavage (100 μl, 250 μg / ml), twice daily for 14 days starting at 11 weeks of age. hj, Compared to disease-free C57BL / 6 control mice. Apc Min / + Strong activation of c-MYC-, N-MYC-, and L-MYC-dependent genes in mice. km,rLon treatment Apc Min / + Suppression of c-MYC-, N-MYC- and L-MYC-dependent genes in mice (n=2 mice per group; fold change FC ≥ 2; P < 0.05 relative to disease-free C57BL / 6 control mice).

[0127] Figure 12 rLon Irf3- / - Inhibition of nephrotoxicity in mice and the degree of similarity between treated and uninfected mice.

[0128] a. Gene expression analysis to identify Escherichia coli CFT073 infection. Irf3- / - Genes associated with nephrotoxicity, kidney injury, or kidney-specific cell death in mice. Strong activation of MYC and IRF7-dependent genes regulating kidney injury or kidney-specific cell death in the placebo group compared to the uninfected group. Red, upregulated; blue, downregulated; fold change FC ≥ 2; P < 0.05 compared to the uninfected group. b, compared to the placebo group, rLon treatment completely suppressed genes regulating kidney injury or kidney-specific cell death. c, compared to placebo, rLon treatment inhibited kidney injury, damage, and cell death pathways. d, except... LyzFurthermore, the top-ranking regulatory nephrotoxic genes in the placebo group were not regulated in the untreated group. e, Principal component analysis of the whole kidney RNA dataset. The right cluster shows uninfected mice (green), and the adjacent clusters show rLon-treated mice (purple). The left cluster shows the placebo group (red). fh, Volcano plots of all detected genes (f), all MYC-dependent genes (g), and all nephrotoxic genes (h) (n = 2 per group; FC vs. uninfected; white, no change; red, activated; blue, suppressed). The placebo group distinguished two gene categories; the first category was those with a strong disease response (n = 3695), and the second category was the remaining genes that were not regulated by the disease (n = 16168). In rLon-treated mice (n = 43), the first category (disease-related genes) was suppressed, but the second category (genes unrelated to the disease) was not significantly regulated by rLon treatment (n = 19820). MYC-dependent genes and nephrotoxic genes showed similar patterns in the rLon-treated group, similar to the uninfected control group.

[0129] Figure 13 , Figure 1 Supplementary data

[0130] Will Irf3- / - Mice were infected with the urinary tract pathogenic Escherichia coli strain CFT073, and kidney tissues were obtained at 24 hours and 7 days for gene expression analysis. Ac, heatmap (a), histogram (b), and Venn diagram (c) compare the gene expression profiles of infected and uninfected mice at 24 hours and 7 days (n=2 per group; red, upregulated; blue, downregulated; fold change FC≥2, P<0.05 compared to uninfected mice). D, Infection Irf3- / - Volcano plot of full gene expression in mice 24 hours later, and further increase in gene expression 7 days later. 2.

[0132] e, 24 hours later, in the infected Irf3- / - In mice, Myc and Myc-dependent gene networks are activated, including the innate immune response network. rLon treatment suppresses the innate immune response to infection (orange, activation; blue, inhibition). Data are for healthy, uninfected mice. Irf3- / - The mice were standardized.

[0133] Figure 14 , Figure 1 Supplementary data

[0134] Compared with the placebo group, c-Myc protein staining was observed in kidney tissue sections from the rLon-treated group. Fluorescence microscopy; representative images are shown below. Figure 1 Green, c-Myc; Blue, cell nucleus; Scale bar, 20 μm; n = 3-4 mice per group.

[0135] Figure 15 , Figure 1 Supplementary data.

[0136] Pathway analysis showed that rLon treatment could inhibit Irf3- / - Interferon signaling, Toll-like receptor signaling, NF-κB signaling, and inflammasome pathways in mice. (n=2 per group; red, upregulated; blue, downregulated; fold change FC≥2, P<0.05 compared with uninfected mice).

[0137] Figure 16 , Figure 2 Supplementary data.

[0138] a, f, k, placebo, or rLon treatment group Irf3- / - Gross appearance of mouse kidneys. be, gj, lo, lower gross pathology, urinary neutrophil count, and bacterial counts in urine and kidney tissue were compared in mice treated with rLon or placebo. Results from three independent experiments are presented. In the first and second experiments, n=5 mice were treated each time; in the third experiment, n=3 mice were treated each time. Lines represent medians (b, c, g, h, l, m), and data were analyzed using the Kruskal-Wallis test and Dunn correction. Data are expressed as mean ± sem (d, e, i, j, n, o) after two-way ANOVA following Bonferr-Loni multiple comparisons test. P<0.05, P<0.01, P<0.001, no statistical significance.

[0139] Figure 17 , Figure 2 Supplementary data.

[0140] Staining whole kidney sections with hematoxylin and eosin (H&E) revealed extensive neutrophil infiltration into the infected kidney and the formation of microabscesses. RLon-treated mice showed no pathological features and had lower neutrophil infiltration (representative images are shown below). Figure 2 As shown; n=4 mice per group; scale bar, 500μm).

[0141] Figure 18 , Figure 2 A control experiment.

[0142] a, b, Effects of rLon on the in vitro growth of *E. coli* CFT073. *E. coli* CFT073 was cultured in Luria broth (black) for 3.5 hours, followed by the addition of 100 mg / ml cefotaxime (red), 250 μg / ml (dark blue), or 500 μg / ml (blue) rLon. OD600 was measured at 0, 1, 2, 3, 4, and 5 hours. rLon had no effect on the growth of *E. coli* CFT073. Each rLon concentration was tested in duplicate as a positive control, along with cefotaxime. c, d, Cell death assays of A498 renal cell carcinomas based on Presto Blue (c) and ATP Lite (d) measurements of full-length rLon and NTD, AAA+, and peptidase domain peptides. No toxicity was detected in full-length rLon, NTD, AAA+, or peptidase domain peptides.

[0143] Figure 19 , Figure 9 Supplementary data.

[0144] a, infection Irf3- / - High-resolution confocal imaging of mouse kidney tissue sections shows strong staining of bacteria and neutrophils, as well as colocalization of pathological areas (n=4 mice per group; red, neutrophils; green, bacteria; blue, cell nuclei). b, an image segmented from a.

[0145] Figure 20 , Figure 4 Supplementary data.

[0146] Compared with the placebo group, N-Myc protein staining was observed in kidney tissue sections from the rLon-treated group. Confocal microscopy; representative images are shown below. Figure 4 Green, N-Myc; Blue, cell nucleus; Scale bar, 20 μm; n = 2-5 mice per group.

[0147] Figure 21 , Figure 4 Supplementary data.

[0148] Compared with the placebo group, L-Myc protein staining was observed in kidney tissue sections from the rLon-treated group. Confocal microscopy; representative images are shown below. Figure 4 Green, L-Myc; Blue, cell nucleus; Scale bar, 20 μm; n = 2-5 mice per group.

[0149] Figure 22 , Figure 5 Supplementary data.

[0150] Analysis of the degradation of N-MYC protein by ac, rLon, peptidase, and AAA+ domains. After 120 minutes, N-MYC (50 ng) was degraded by rLon (0.5 or 1 μg, a), and in the presence of the AAA+ domain peptide, by the rLon peptidase domain (b). N-MYC protein controls show the presence and absence of incubation. This effect was reversed in the presence of a protease inhibitor (b). d, Purity of recombinant Lon, N-terminal domain (NTD), AAA+, and peptidase domains, verified by Coomassie staining. eg, Western blot analysis showing the uptake of Lon and peptides with different domains in different cellular components.

[0151] Figure 23 , Figure 5 Supplementary data.

[0152] a. Western blot analysis of Lon degradation of c-MYC in whole-cell lysates and nuclear extracts. b. Western blot analysis of rLon degradation of N-MYC in whole-cell lysates and nuclear extracts. c. Western blot analysis of the degradation of L-MYC by rLon and different domains in whole-cell lysates and nuclear extracts.

[0153] Figure 24 , Figure 6 Supplementary data.

[0154] Biacore data showed a dose-dependent interaction between full-length Lon and NTD peptides and His-labeled c-Myc and N-Myc captured on the sensor chip NTA. Cells without nickel loading served as a reference (blue) for nonspecific binding of proteins and chemicals, while buffer-only (green) served as a control. Histidine-labeled ligands (c-Myc or N-Myc) were injected onto the nickel-activated sensor surface for capture. Full-length Lon or NTD peptides were then injected onto the captured His-labeled ligands using five different dilutions to monitor the interaction between the sample and the His-labeled ligands (magenta). Detailed Implementation

[0155] Example

[0156] Example 1

[0157] method

[0158] strain

[0159] The prototype APN strain of *Escherichia coli* CFT073 (O6:K2:H1) (reference) was cultured on trypsin-soybean agar (TSA) plates (16 h, 37 °C) and then in phosphate-buffered saline (PBS, pH 7.2, 10⁻⁶). 10 Harvest at (CFU / mL) and dilute appropriately. Static culture of E. coli CFT073 overnight in Luria broth for animal inoculation.

[0160] Experimental urinary tract infection

[0161] Mice were housed at the MIG animal facility in Lund, Sweden, in individually ventilated cages (IVCs) free from specific pathogens, under a 12-hour light-dark cycle at a constant temperature of 23°C. Lights were turned on at 7:00 AM, and food and water were freely available. Female C57BL / 6 wild-type mice aged 9-15 weeks were used. Irf3- / - and Irf7- / - And mice. The C57BL / 6 background was provided by the Riken Biological Resource Center in Japan with permission from *T. Taniguchi*. Irf7- / - and Irf3- / - Mice.

[0162] Mice anesthetized with isoflurane were inoculated intravesically with Escherichia coli CFT073 (0.1 mL containing 10... 8 CFU infection was detected, and the kidneys were euthanized under anesthesia. Kidneys were aseptically removed, and macroscopic pathology was documented by photography. Scoring was based on the degree of congestion, edema, and abscess, ranging from 0 to 10, where 0 was similar to the uninfected control group, and 10 represented the most severe congestion, edema, and abscess. One kidney was isolated for RNA extraction (on dry ice) and immunostaining (in OCT compound or 10% (v / v) formalin). Viable cell counts were determined by growth on TSA plates (37°C, overnight) after appropriate serial dilution. Neutrophils in uncenterable urine were counted using a hematology counter.

[0163] colon cancer model

[0164] C57BL / 6J- Apc Min / J mice developed spontaneous intestinal adenoma formation and were housed in a laboratory medicine department at 11 or 20 weeks of age. Mice were treated twice daily for 14 days with oral gavage (200 μl) of rLOn protease (250 μg / mL). At sacrifice, the intestines were aseptically harvested, and a segment of the intestine was prepared for mRNA extraction for further analysis.

[0165] In vivo bacterial Lon protease treatment

[0166] C57BL / 6 Wild Type Irf3- / - and Irf7- / - Mice were treated with recombinant bacterial Lon protease via intraperitoneal injection (100 μL, 250 μg / mL) once daily for 7 days, starting from the day before day 0, the day before E. coli O73 bladder infection. Mice were sacrificed 24 hours and 7 days later. Sham-treated mice received PBS.

[0167] Whole genome transcriptomics analysis

[0168] Total kidney RNA was isolated using the RNAeasy Mini Kit (Qiagen), amplified using the GeneChip 3'IVT Express Kit (Affymetrix), hybridized to a 430 PM mouse genome array, and internally scanned using the GeneAtlas system (Affymetix). Data were normalized using Robust Multi-Average implemented in the transcriptome analysis console software (v.4.0.1.36, AppliedBiosystems). Significantly altered genes were counted by comparing bacteria-infected cells or tissues with uninfected controls (PBS control group) and ranked by relative expression (using a two-way ANOVA model with moments, absolute fold change >1.41, 1.5, or 2.0). Heatmaps were constructed using GraphPad (Prism v.9). Differentially expressed genes were functionally characterized using ingenuity pathway analysis software (IPA, Qiagen).

[0169] Immunohistochemistry

[0170] Frozen tissue sections (8µm, Leica microtome) embedded and frozen in OCT were collected on positively charged microscope slides (Superfrost / Plus; Thermo Fisher Scientific), fixed in acetone-methanol (1:1, 10 min), dried, infiltrated (0.2% Triton X-100, 5% normal goat serum / PBS), and stained with rat anti-neutrophil primary antibody [NIMP-R14] (1:200; Abcam, ab2557), rabbit monoclonal anti-c-Myc antibody (1:100; ab32072, Abcam), L-Myc, and N-Myc antibodies. The tissues were then stained with rabbit anti-rat and goat anti-rabbit IgG secondary antibodies labeled with Alexa 488 or Alexa 568 (molecular probes, A-21210, A-11001, and A-11011). Cell nuclei were counterstained with DAPI (0.05 mM; Sigma-Aldrich). The slides were examined using a fluorescence microscope (AX60, Olympus Optical). Hematoxylin staining solution (Richard-Allan Scientific Signature Series Hematoxylin 7211) and Eosin-Y 7111 (Thermo Scientific) were used for counterstaining the tissue sections.

[0171] Human renal epithelial cells

[0172] A498 human kidney epithelial cells (A498, ATCC HTB-44) were cultured overnight in RPMI-1640 supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, and 10% fetal bovine serum (FBS) in 6-well plates (for Western blotting) or 8-well slides (for confocal imaging) at 37°C, 90% humidity, and 5% CO2. The cells were then infected with bacteria from fresh, serum-free RPMI.

[0173] siRNA transfection

[0174] Following the manufacturer's instructions, A498 human renal epithelial cells were transfected with siRNA using Lipofectamine® RNAiMAX reagent. Knockout efficiency was verified by qRT-PCR. After 48 hours of incubation, transfected cells were harvested for protein extraction or confocal microscopy examination.

[0175] Immunofluorescence

[0176] A498 cells were fixed (3.7% formaldehyde, 10 min), permeabilized (0.25% Triton X-100, 5% FBS, 15 min), blocked (5% FBS, room temperature, 1 h), and incubated overnight at 4°C in 5% FBS with anti-Myc (ab32072, Abcam), anti-IRF7 (Abcam), anti-phosphate-IRF3, or anti-total IRF3 primary antibody (1:200). Cells were then incubated at room temperature for 1 h with Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (1:2000; A-11034, Life Technologies). After nuclear staining (DRAQ5, Abcam), the cells were mounted (Fluoromount, Sigma-Aldrich), imaged using a laser scanning confocal microscope (LSM510 META confocal microscope, Carl Zeiss), and quantified using ImageJ software 1.46r (NIH).

[0177] Protein blot

[0178] Cells were cultured in 6-well plates (4 x 10⁻⁶). 5 Cells / well (Thermo Fisher Scientific), washed with ice-cold PBS, lysed with RIPA buffer containing protease and phosphatase inhibitors (Roche Diagnostics). Cell lysates were run on SDS-PAGE (4-12% Bis-Tris gel, Invitrogen) and blotted onto PVDF membranes (GE Healthcare), blocked with 5% bovine serine albumin (BSA), and incubated with anti-Myc (ab32072, Abcam), anti-IRF7 (Abcam), anti-phospho-IRF3 or anti-total IRF3 primary antibody (1:1000) and primary antibody (1:1000 in 5% BSA). Blots were washed with 0.1% PBS (PBST), incubated with secondary antibody in 5% skim milk powder or BSA (1:4000, goat anti-rabbit HRP, Cell Signaling), washed again with PBST, developed with ECL Plus assay kit (GE Healthcare), and analyzed using Bio-RAD ChemiDoc. TM System imaging. β-actin (1:4000, A5441, Sigma) was used as a loading control.

[0179] Quantitative and statistical analysis

[0180] For data determined to follow a Gaussian distribution (D'Agostino and Pearson normality tests), statistical differences were determined using a two-tailed t-test or a one-way ANOVA with Greenhouse-Geisser correction. For nonparametric data, a two-tailed Mann-Whitney U test or a Kruskal-Wallis test with Dunn correction was used, and paired data were analyzed using the Wilcoxon paired signed-rank test. Statistical significance was determined using GraphPad (Prism v.9), with a significance level set to [value missing]. P<0.05 P<0.01 and P < 0.001. All data are expressed as mean, and all error bars represent standard deviations. Sample size (n) represents the number of independent experiments or the number of mice used in the study.

[0181] result

[0182] Susceptible Irf3- / - Rapid MYC response in mice to infection

[0183] MYC transcription factors define fundamental aspects of kidney growth and development. In childhood, acute pyelonephritis is associated with renal scarring, impaired kidney growth, and persistent detrimental effects on kidney function. Female Irf3- / - mice were infected with the urinary tract pathogenic Escherichia coli strain CFT073 (… Figure 1 Mice infected via intravesical injection developed severe kidney disease seven days later, with elevated pathological scores characteristic of acute pyelonephritis. Bacterial counts remained high seven days later, and histopathology showed evidence of neutrophil infiltration and abscess formation in the kidney tissue compared to uninfected mice. Figure 2 ).

[0184] Gene expression analysis of renal tissue RNA detected a rapid response of C-MYC to infection. After 24 hours, total gene expression in infected mice was elevated (1093 significantly regulated genes), and the MYC-dependent gene network and gene expression remained activated after 7 days, including MYC and MYC-dependent genes (fold change FC>2, P<0.05 compared to the uninfected control group). Compared to the uninfected control group, the response of C-MYC to infection was significantly higher. Irf3- / - Immunohistochemistry of mouse tissue sections detected a parallel increase in renal C-MYC protein levels. C-MYC staining was higher along the inner lining of the renal pelvis mucosa and the renal papilla, consistent with local MYC reactions at the site of infection. Figure 2Compared with healthy controls, the innate immune C-Myc-dependent regulator was strongly upregulated in Irf3- / - mice. Figure 1 g), including toll-like receptor NFkB, IL-6 and type I interferon, as well as IL-8 and S100A8 that activate neutrophil recruitment.

[0185] The results showed that a strong disease-associated C-Myc response was present in the kidneys of infected Irf3- / - mice.

[0186] rLON treatment inhibited MYC overexpression in infected mice.

[0187] The inventors then investigated whether rLon acted as a MYC inhibitor in infected Irf3- / - mice and whether rLon treatment had protective potential. Irf3- / - mice were intraperitoneally injected with recombinant Lon protease (rLon, 250 μg / ml) once daily for seven days, starting the day before infection. Figure 1 a). The placebo group received PBS. rLON treatment reduced the total number of regulated genes from 3696 in the placebo group to 43 in the rLON-treated group (fold change ≥2 on day 7, P < 0.05). Figure 1 d). C-Myc overexpression was almost eliminated, as evidenced by a reduction of 747 dysregulated Myc genes in the placebo group to only two genes in the rLon treatment group (day 7, fold change FC≥2, P<0.05). Figure 1 (b, d). Compared with the placebo group, immunohistochemical staining of C-MYC in kidney tissue sections from infected Irf3- / - mice treated with rLon was reduced in parallel, confirming the therapeutic effect at the protein level. Figure 1 e, f).

[0188] rLon treatment significantly reduced the innate immune response to infection. This included a network of MYC-dependent regulators of the innate immune response, which was strongly suppressed in rLon-treated Irf3- / - mice. Major innate immune signaling pathways activated in the placebo group were no longer regulated in treated mice, including Toll-like receptors, IL-6-, IL-8-, TNFR, NF-κB, and inflammasome signaling (fold change FC≥2, P<0.05). Figure 1 When hIrf7-dependent genes are suppressed, interferon signaling is severely weakened.

[0189] RLon-treated mice were protected from severe acute kidney disease.

[0190] rLon treatment further demonstrated that it could prevent infected Irf3- / - mice from developing acute kidney disease. Figure 2Compared with the placebo group, rLon treatment inhibited the development of gross pathology ( ). Figure 2 a, b), reduced the number of bacteria in urine and infected kidneys ( Figure 2 c, d). Neutrophil infiltration and abscess formation were suppressed, reducing the neutrophil count in the urine ( Figure 2 e). Seven days later, compared with the placebo group, the renal tissue structure in the rLon treatment group remained largely intact. Figure 2 fh).

[0191] High-resolution confocal imaging of mouse tissue sections infected with rLon further investigated the differences between the rLon treatment group and the placebo group compared to the placebo group. Figure 2 The site of infection was identified by the significant co-localization of bacteria and neutrophils, indicating abscess formation. Strong staining of bacteria and neutrophils was detected in lesions in the inner mucosa of the renal pelvis, renal papillae and collecting ducts, and near the apex of the renal pelvis. Figure 2 i). This staining pattern was not observed in rLon-treated mice, where bacterial and neutrophil staining was low or undetectable (i). Figure 2 j, k).

[0192] The results showed that rLon treatment could inhibit infection-induced MYC dysregulation and prevent the development of acute kidney disease in susceptible mice.

[0193] Mice treated with rLon were protected from kidney damage.

[0194] Gene expression analysis is used to examine kidney tissue damage caused by infection in more detail. Figure 3 Compared to uninfected mice, the placebo group showed activation of numerous genes associated with kidney injury, consistent with the widespread kidney pathology observed in these mice. Figure 3 a). In contrast, the rLon treatment group showed almost no renal injury response, with only one significantly regulatory gene associated with nephrotoxicity (n=1), and no evidence of a broader MYC-dependent response. Figure 3 b). A large number of kidney injury-related genes in the placebo group could be defined as MYC-related (34%), including a subset of IRF7-dependent genes (NF-κB, STAT3, FOS, and IRF). Another subset of kidney injury-related, IRF7-related, but MYC-independent genes included TLR, MYD88, and CD40. Figure 3 a).

[0195] The results showed that MYC dysregulation led to nephrotoxicity in infected mice, including genes associated with nephrotoxicity, kidney injury, or kidney-specific cell death. rLon treatment prevented kidney injury by targeting and inhibiting this response.

[0196] rLon treats targeted dysregulation of MYC

[0197] The strong protective effect of rLon treatment against infected kidneys raises the question of whether rLon primarily targets MYC dysregulation associated with disease in infected kidneys. Principal component analysis of kidney tissue RNA was used to evaluate the entire RNA datasets in the treatment and placebo groups and compared with healthy, uninfected mice. The placebo group formed a cluster far removed from healthy mice compared to the rLon treatment group, which was very similar to uninfected mice. Figure 3 f).

[0198] The effect of rLon on MYC dysregulation in infected kidneys was further analyzed by directly comparing the placebo group and the rLon treatment group with healthy, uninfected mice. Figure 3 e). rLon treatment was shown to specifically suppress disease-related responses, a characteristic of the placebo group, while preserving homeostatic MYC expression to a degree similar to that in healthy, uninfected mice.

[0199] Similar analyses were performed on Myc-dependent gene subsets in these datasets. The results showed that rLon treatment primarily targets dysregulation of MYC associated with disease in infected kidneys.

[0200] rLon treatment inhibits the L-MYC and N-MYC response to infection.

[0201] The C-MYC, L-MYC, and N-MYC family members are functionally similar but exhibit different expression patterns and tissue specificity. L-MYC is localized to the renal pelvic mucosa and papillae, and in this study, the infection was located in the papillae. N-MYC is also expressed during kidney development. Therefore, the inventors investigated whether the kidney's response to infection also includes N-MYC and L-MYC, and whether rLon treatment affects L-MYC and N-MYC levels.

[0202] Compared to uninfected controls, mice infected with Irf3- / - showed strong activation of N-Myc and L-Myc expression at 24 hours and 7 days. In the placebo group, the activated N-Myc gene network comprised approximately 25% of all genes defined by IPA as N-MYC-related. Figure 4 A similar activation pattern of L-MYC was detected, including approximately 33% of genes that could be defined as L-MYC-related. Immunohistochemistry confirmed increased levels of N-MYC and L-MYC protein in kidney tissue sections, with increased L-MYC staining detected along the renal pelvis and renal papillae 24 hours and 7 days after infection with Irf3- / - mice. Figure 4 c, f).

[0203] rLon treatment further demonstrated inhibition of L-Myc and N-Myc responses to infection in Irf3- / - mice. Compared to the placebo group, L-Myc expression was reduced by 95% (number of genes). Figure 4 (b, e) After 24 hours, only one L-MYC-dependent gene showed differential expression, and after 7 days, no gene remained activated (fold change FC>2, p<0.05). Compared with 400 genes in the placebo group, only two N-MYC-related genes showed differential expression after 7 days of rLon treatment. The treatment was also accompanied by loss of L-MYC and N-MYC staining in rLon-treated mouse tissues (Fig. c, f).

[0204] The results showed that N-Myc and L-Myc were dysregulated in the kidneys of infected Irf3- / - mice. In addition to targeting C-Myc, rLon treatment also targeted N-Myc and L-Myc responses, which were suppressed.

[0205] rLon treatment inhibited the expression of N-Myc and L-Myc in a colorectal cancer model.

[0206] Oral treatment with rLon has previously been shown to suppress Apc, a predisposition to colon cancer. Min / + c-MYC expression in mice can prevent tumor progression and mortality. Here, we examined intestinal slices to investigate whether the expression of N-Myc and L-Myc was increased in mice with intestinal tumors. Figure 4 h, m). Gene expression analysis detected that, compared with the healthy C57BL / 6 control group, the placebo group showed significant activation of N-Myc and L-Myc expression in tumor biopsy tissues, and Apc carrying tumors. Min / + N-Myc and L-Myc-dependent gene expression networks were activated in mice.

[0207] In contrast, compared to the healthy C57BL / 6 control group, Apc in rLon treatment... Min / + In intestinal tissue sections from mice, the expression of N-Myc and L-Myc was not activated. Compared with the placebo group, rLon-treated Apc... Min / + Gene expression in mice was significantly reduced.

[0208] The results indicate that rLon is a pan-MYC inhibitor in bacterial infection and cancer models.

[0209] Cellular interactions between rLon and rLon peptide domains

[0210] Lon proteases are responsible for protein quality control in various organisms, including bacteria, where they hydrolyze unfolded and misfolded proteins. Furthermore, Lon has been shown to interact with DNA. The role of Lon in a kidney infection model supports the correlation between these two mechanisms, as treatment in mice resulted in decreased levels of MYC protein and a strong suppression of MYC-dependent gene expression. Similar to its effects on non-mammalian proteins, the Lon protease domain may be responsible for the degradation of MYC proteins, while the second domain is responsible for transcriptional repression.

[0211] To perform these effector functions, Lon proteins enter human cells and interact directly with MYC protein isoforms or transcriptional mechanisms through their domains. To identify Lon protein domains involved in MYC repression, we recombinantly expressed globular Lon peptide domains and exposed human renal cell carcinoma cells to full-length rLon protein, N-terminal domain peptides (NTD, residues 1-245), ATPase domain peptides (AAA, residues 246-584), or peptidase domain peptides (residues 585-784). Figure 5 a). As shown in live-cell confocal imaging, Alexa Fluor-labeled full-length proteins and peptides were effectively internalized and detected in the cytoplasm, perinuclear region, and nuclear region of the cell. Figure 5 c). Western blot analysis of whole-cell extracts confirmed the uptake of Lon and peptides ( Figure 5 b). Through subcellular grading, peptides are further localized to different compartments.

[0212] Analysis of subcellular components of cells treated with full-length protein or each peptide demonstrated a reduction in L-MYC, N-MYC, and C-MYC. The decrease in MYC levels primarily affected nucleoprotein levels (…). Figure 5 (d, f). The full-length Lon affects all three types of MYC: N-terminal domain L-MYC, AAA domain L-MYC, peptidase domain N-MYC, and L-MYC.

[0213] The effects of Lon and peptide domains on MYC gene expression were further investigated by qRT-PCR using C-MYC, L-MYC (isomers I and II), or N-MYC-specific primers. Figure 5 g). In human renal cell carcinoma cells exposed to either the N-terminal domain peptide or the full-length Lon protein, L-MYC expression was significantly reduced. A significant but weak effect of the N-terminal domain (NTD) was observed in N-MYC. However, C-MYC was not significantly affected. The peptidase domain showed a significant inhibitory effect on L-MYC expression, indicating that L-MYC is targeted more extensively at the transcriptional level than C-MYC.

[0214] Computer simulations were used to further investigate the Lon domain and its peptide-targeted MYC domain.

[0215] AlfaFold was used to predict interactions with C-MYC, N-MYC, and L-MYC. Figure 5 (h, i). Successful computer-generated docking predictions showed that the C-terminal portion of C-MYC (amino acids 356–439) interacts with LON NTD, with an IPTM score of 0.697. Furthermore, there was clear indication that the C-terminal portion of N-MYC (amino acids 354–464) interacts with Lon in a similar binding pattern and with a similar IPTM score. The targeting MYC sequence corresponds to a zipper domain rich in α-helical leucine, which binds DNA and regulates transcription, particularly the expression of MYC-dependent genes. L-MYC is not targeted by NTDs.

[0216] The results indicate that NTD regulates cellular MYC levels, partly by binding to a zipper domain rich in α-helicoleleucine.

[0217] Disease specificity of MYC response and rLon protease treatment

[0218] To better understand the consequences of Lon's inhibition of MYC, a whole dataset of infected mice (7 days) was compared between the placebo group and Lon-treated mice. Healthy, uninfected mice served as controls. Principal component analysis (PCA) showed that Lon-treated mice formed a cluster near healthy mice. In contrast, the placebo group formed a cluster far from both the Lon-treated group and healthy mice (90% variance, first principal component), indicating that the gene expression profiles of healthy and Lon-treated mice were similar and that the expression of disease-related genes was suppressed.

[0219] To further investigate this hypothesis, a heatmap was constructed based on the total number of genes detected (n=19863). Only a distinct set of upregulated or downregulated genes was detected in the placebo group, suggesting a disease association. LON-treated mice mirrored the gene expression patterns of healthy mice, strongly supporting the conclusion that LON treatment primarily suppresses disease-related MYC responses, including C-MYC, N-MYC, and L-MYC.

[0220] The mechanism by which Lon and Lon domains suppress MYC

[0221] To investigate the mechanism by which Lon inhibits MYC, the full-length Lon protein and each globular domain were purified using a C-terminal Strep tag. Figure 5 a, b). Through ATP and Mg 2+The direct degradation of MYC by Lon was quantified by mixing rLon with recombinant c-MYC in the presence of PBS. Compared with PBS, after incubation with rLon at 37°C for 120 minutes, Western blot analysis showed a significant decrease in c-MYC protein levels. Figure 5 c). Using the same protocol, the Lon peptidase domain significantly reduced c-MYC protein levels, and this effect was enhanced in the presence of the ATP-binding AAA+ domain, which is essential for complete peptidase activity in bacteria. 26 ( Figure 5 d). In the presence of protease inhibitors, degradation is inactivated ( Figure 5 e).

[0222] To investigate whether Lon proteases can target intracellular MYC, human renal cell carcinoma cells were treated with full-length rLon or each Lon domain peptide, and visualization and quantification were performed using AZ647 fluorescent labeling. Live-cell imaging detected cellular uptake of rLon (…). Figure 5 f), rLon staining was evident in the cytoplasm and nucleus. Each peptide labeled with AZ647 showed a similar uptake pattern ( Figure 5 f), this was also confirmed by Western blot analysis of whole-cell extracts and nuclear extracts from cells exposed to rLon or each peptide. Figure 5 g).

[0223] To quantify the effect of rLon treatment on cellular MYC protein levels, Western blot analysis was performed on whole-cell extracts. Figure 5 h, i). In cells exposed to full-length rLon, c-MYC protein levels were significantly reduced, with this effect being particularly pronounced in nuclear extracts compared to cells treated with PBS. However, c-MYC levels were not affected by individual peptide domains (h, i). Figure 5 h). rLon treatment reduced L-MYC levels, including isoform II (20 kDa) and isoform I (40 kDa), and was strongly influenced by NTD, AAA+, and peptidase domains, although these dissociated domains primarily affected the shorter isoforms (h). Figure 5 h, i). Cells treated with full-length rLon or peptidase domains from whole-cell lysates and nuclear extracts showed significantly reduced N-MYC protein levels after rLon treatment. Figure 5 h).

[0224] Using specific primers for MYC, MYCL (isomers I and II), or MYCN, the significant inhibition of MYC expression in rLon-treated cells was investigated by qRT-PCR. Figure 5i). Compared with control cells, MYCL expression was strongly suppressed in human kidney cells exposed to full-length protein (FC-9.7), and further suppression was detected in cells exposed to NTD peptide (FC-38.4) and peptidase domain peptide (FC-21) compared with PBS-treated control cells. In contrast, rLon or Lon domain peptides did not significantly regulate MYC expression (FC<2) compared with PBS-treated control cells, and MYCN expression was weakly regulated (FC-2.1 and -2.7).

[0225] Lon targets MYC-MAX dimerization and NTD domain peptides to affect MYC-dependent gene expression.

[0226] Effective suppression of MYC-dependent gene expression in the disease model suggests the possible involvement of other mechanisms. Therefore, the rLon-targeted MYC protein domain was identified using computer prediction techniques. Figure 6 ad). Using the AlphaFold multimer assay, the interactions of the Lon NTD peptide with the C-terminal portion of C-MYC (amino acid positions 356-439) and N-MYC (amino acid positions 354-464) were predicted, with an iPTM score of 0.697. Figure 6 a, b). The c-MYC and N-MYC sequences predicted to be targeted by Lon's NTD correspond to the MAX binding sites in the leucine zipper domain. Biacore analysis showed that the full-length Lon protein and NTD peptide bind in a dose-dependent manner to His-labeled c-MYC (full-length), c-MYC (C-terminal domain), and N-MYC (full-length), K D Within the μM range, this indicates that the full-length Lon protein and NTD peptide have affinity for both c-MYC and N-MYC. Figure 6 c). The structural superposition of the crystal structure with that of the MYC-MAX complex shows that the NTD peptide and MAX almost completely overlap. Figure 6 d), indicating that NTD targets the MAX binding site ( Figure 6 d), indicating that NTD targets the MAX binding site ( Figure 6 (d) This is a mechanism by which Lon replaces MAX and reduces the formation of the MYC-MAX complex, which may explain the significant reduction in MYC-dependent gene expression observed in in vivo models after Lon treatment.

[0227] This hypothesis was addressed using the ELISA-based TransAM c-MYC assay, which quantifies the binding of c-MYC to its shared E-box DNA sequence. The rLon protein and its NTD and AAA+ domains reduced c-MYC binding to DNA. Figure 6e). The function of the NTD domain was consistent with computer-predicted NTD-MAX binding sites, and the function of the AAA peptide was consistent with the known affinity of this domain for DNA. The effects of Lon on MYC-dependent gene expression were further investigated in cells exposed to rLon, NTD, or AAA+ peptides. Genome-wide transcriptome analysis identified MYC targets that were significantly inhibited in human renal cell carcinoma cells treated with NTD and AAA+ peptides, consistent with in vitro data on MYC-MAX inhibition, while the effect of full-length Lon was weaker. Figure 6 g, h).

[0228] The results indicate that the MYC transcription factor family is a target of bacterial Lon proteases, which are absorbed by human cells and affect cellular MYC levels in multiple ways. First, MYC degradation, jointly executed by the peptidase domain and the ATP-bound AAA+ domain, was detected in vitro. Second, the decrease in c-MYC, N-MYC, and L-MYC levels after rLon treatment was attributed to the degradation of MYC by the peptidase and AAA+ domain, leading to the inhibition of MYC expression. Finally, by targeting the MAX binding site, the Lon NTD domain was shown to inhibit MYC-MAX heterodimerization, which is crucial for DNA binding to the helical-loop-helical domain that subsequently initiates MYC-dependent transcription. 27 .

[0229] discuss

[0230] MYC dysregulation is associated with severe disease states, most broadly defined as the C-MYC proto-oncogene and its role in cancer. Selective inhibitors targeting dysregulated MYC would ideally affect most human cancers, but such inhibitors are difficult to find. This study identified Lon, a bacterial-derived protein, as a pan-MYC inhibitor with potent effects in reversing disease-associated MYC dysregulation in infection and cancer models. The therapeutic effect was associated with broad suppression of the MYC-dependent gene network and loss of overexpression of C-MYC, N-MYC, and L-MYC. These effects are presented here to reflect the different rLon protein domains that either increase MYC degradation or inhibit the expression of MYC and MYC-dependent genes, thereby reducing pan-MYC levels. These results suggest that Lon is a novel and powerful tool for neutralizing the destructive effects of MYC dysregulation across various disease states.

[0231] The rLon protease has previously been shown to degrade c-MYC in an in vitro, time-dependent manner and reduce c-MYC levels in human cells and cancer models. In this study, these effects were extended to include N-MYC and L-MYC, which exhibit disease-associated hyperactivation and sensitivity to rLon similar to those of C-MYC. Structural-functional analysis revealed two repressive mechanisms. A synthetic peptide containing the Lon protease domain was shown to enter human cancer cells and reduce C-MYC, N-MYC, and L-MYC levels in the cytoplasm and nucleus. Furthermore, a synthetic peptide containing the N-terminal domain was shown to translocate to the nucleus of human cells and act as a potent transcriptional inhibitor, primarily suppressing the expression of L-MYC, N-MYC, and C-MYC. Thus, in addition to degrading C-MYC, the Lon peptidase domain also reduces the levels of N-MYC and L-MYC. The role of rLon is further extended to include transcriptional repression performed by the N-terminal domain and primarily affecting L-MYC expression.

[0232] The MYC protein family shares a basic helical-loop-helical leucine zipper structure, with significant structural homology among conserved C-terminal regions. The helical-loop-helical leucine zipper motif is crucial for DNA binding of MYC transcription factors at transcription initiation sites in many MYC-dependent promoters. In this study, the helical-loop-helical leucine zipper domain was identified as a target of the Lon N-terminal domain. A direct effect on MYC gene expression was detected, but in addition, the MYC-dependent gene network was strongly repressed, leading to widespread transcriptional repression. A valid alignment of the Lon NTD with the helical-loop-helical leucine zipper motif may contribute to this broad effect by inhibiting MYC dimerization required for DNA binding.

[0233] MYC is widely expressed during tissue development and overexpressed in a variety of tumors. MYCN is similarly regulated and dysregulated in different cancer types, and N-MYC can functionally replace C-MYC in the context of cell growth and differentiation. Despite structural similarities, they exhibit different expression times and tissue specificity. Although MYCL also plays a role in tumor and tissue development, it has been reported to be crucial for embryonic development, and MYCL expression initiates T cells in response to viral and bacterial infections. Tissue expression of MYCL, including in the kidneys, is more restricted. These findings identify these regions as targets for bacterial kidney infection, with high-resolution imaging revealing abscesses containing bacteria and neutrophils in sham-treated mice but not in rLon-treated mice. This is consistent with the role of L-MYC overactivation in kidney pathology and the protective effect of rLon against nephrotoxicity and tissue damage in these sites.

[0234] Many methods for inhibiting MYC have proven unsuccessful due to the lack of suitable pockets for binding low molecular weight inhibitors and the absence of catalytic activity. Furthermore, its nuclear localization makes MYC more difficult to target with therapies such as monoclonal antibodies. Full-length Lon proteases, peptidases, and NTD domains have been shown to reach tumor cell nuclei and effectively reduce nuclear C-MYC, N-MYC, and L-MYC protein levels. Methods for inhibiting MYC also include inhibiting transcription and translation by targeting the obligate MYC interacting chaperone MAX, modulating MYC stability, targeting upstream pathways of MYC, or blocking downstream genes exhibiting synthetic lethality when MYC is overexpressed. This mechanism may be unrelated to rLon, as rLon proteases exhibit selectivity for c-MYC, and MYC chaperones MAX, CEBP-δ, AKT, CDK9, CPEB1, GRP75, actin, or histone H3 are also involved. 8 No degradation. OmoMYC microprotein, corresponding to the HBLH domain of MYC, exerts its effects by blocking the binding of all three forms of MYC to their target promoters and has shown anticancer activity with minimal side effects in a range of preclinical models. OmoMYC is thought to guide MYC to specific subsets of target genes, and its inhibition promotes apoptosis by interfering with the binding of MYC / MAX to the E-box. Lon proteases have a broader effect on gene expression, preferentially inhibiting MYC-related dysregulation. Furthermore, Lon proteases have been shown to strongly inhibit L-MYC expression, likely due to the degradation of C-MYC and N-MYC and the resulting inhibition of L-MYC expression.

[0235] Ideally, therapeutic interventions targeting a specific disease should maintain health without adversely affecting tissue homeostasis. In this study, healthy mice treated with rLon showed no evidence of disease or significant changes in MYC expression, suggesting that Lon may not target normal MYC expression as readily as disease-associated MYC responses. The molecular basis for this apparent specificity is unclear, but several possibilities can be discussed. First, the rLon protease could target disease-specific modifications of C-MYC, optimizing Lon binding and the degradation of its target. MYC dysregulation during cancer and infection may produce structural changes, such as specific phosphorylation patterns, enhancing peptide domain binding and promoting MYC degradation. Second, MYC overexpression may enhance target presentation or its cellular localization, making the protease more effective at its proteolytic function. Third, access to specific MYC promoters by rLon or its N-terminal domains may be enhanced in the open chromatin of rapidly dividing cancer cells, potentially causing significant transcriptional repression detected in disease but absent in healthy tissues.

[0236] Example 2

[0237] method

[0238] Expression and purification of recombinant Lon protease

[0239] Nucleotide sequences of different domains of the Lon protease were chemically synthesized (Genscript), including the N-terminal domain (NTD), ATPase domain (AAA+), peptidase domain (Pep.), and the full-length Lon protein. These genes were cloned using the pASK-63A+ vector (IBA) and labeled with Strep-tag II, which carries the β-lactamase gene (producing ampicillin resistance as a selection marker) and a tetracycline promoter / operon system for tightly controlling its expression. Four different pASK-63A+ expression constructs containing different Lon protease domains were then transformed into competent *E. coli* BL21(DE3) cells using a heat shock method. Selected single colonies were then inoculated into Luria Bertani (LB) medium containing 100 mg / ml ampicillin and cultured at 37°C with shaking at 200 rpm until the optical density (OD600) at 600 nm reached approximately 0.5. Cell pellet was then collected by centrifugation at 14000 xg for 10 minutes. The pellet was disrupted by sonication on ice with lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl) for approximately 10 minutes, with 20-second shocks followed by 40-second intervals between shocks. The pellet was then centrifuged (46000 xg, 30 minutes, 4°C). Purification was performed using a 0.8 cm diameter poly-prep column (Bio-Rad) packed with Strep-Tactin Sepharose (IBA Lifesciences) according to the manufacturer's instructions. The pellet was then purified using elution buffer (50 mM Tris pH 7.4, 100 mM NaCl).

[0240] Lon protease was collected from the column using 5 mM dethiobiotin (pH 7.4) and dialyzed using Snakeskin pleated dialysis tubes (10000 MWCO) in a buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, and 10% glycerol, with gentle shaking at room temperature for at least one hour, followed by overnight incubation at 4°C. The eluted fraction was used immediately or stored at -80°C.

[0241] Computer prediction of MYC-Lon interaction

[0242] AlphaFold Multimer Solution 43The Lon sequences used are identical to those used in the recombinant globular domains. The MYC sequences used are as follows: UniProt ID: P01106-1 (c-MYC), P04198 (N-MYC), and P12524-1 (L-MYC). These sequences were either full-length or fragmented into overlapping segments to ensure that the flexibility of the MYC protein did not mask potential binding sites in the global Lon domain. The sequences were then submitted to CoLabFold using the AlphaFold2 multimer protocol. For searches using sequence fragments, all predictions underwent initial visual plausibility checks (Pymol open source), and complexes were filtered out by purifying the Lon domain with Strep tags or filtering out MYC ends generated from sequence fragmentation. The hits were then ranked according to their respective iPTM scores.

[0243] Surface Plasmon Resonance Analysis

[0244] Real-time biomolecular interaction analysis was performed using a BiacoreX100 instrument (Cytiva Life Sciences, Uppsala, Sweden) equipped with a Sensor Chip NTA (BR100034, Cytiva Life Sciences) and surface plasmon resonance (SPR) technology.

[0245] All experiments were performed at 25°C at a constant flow rate of 30 μl / min using 0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA, and 0.5% v / v surfactant P20 (BR100826, Cytiva Life Sciences). Nickel-free cells were used as a reference for nonspecific binding of proteins and chemicals. The Biacore X100 control software was programmed to first start the instrument with run buffer (0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA, and 0.5% v / v surfactant P20). NiCl2 was introduced to saturate the NTA with nickel ions, followed by washing with 3 mM EDTA. Histidine-labeled ligands were prepared in a running buffer at a concentration of approximately 0.2 μM and injected onto a nickel-activated sensor surface to capture His-labeled ligands, full-length c-MYC (CSBEP015270HU, Cusabo), C-terminal c-MYC (TP762404, Origene), or full-length N-MYC (AP72694, Signalway). Samples were injected onto the captured His-labeled ligands using five different dilutions to monitor the interaction between the samples and the His-labeled ligands. Finally, the chip was regenerated by injection of 350 mM EDTA. Binding affinity was calculated based on the rate constant obtained by fitting the data to a 1:1 Langmuir binding model.

[0246] DNA binding ELISA

[0247] Whether Lon inhibits c-MYC activation was assessed using the TransAM c-MYC kit (43396, active motif). The recombinant c-MYC / MAX complex (81087, active motif) was used as a standard. Recombinant Lon and a single domain were added to 5 or 2.5 ng of the c-MYC / MAX complex, and DNA-bound MYC was quantified according to the manufacturer's instructions.

[0248] Cell culture

[0249] A498 human renal cell carcinoma cells (ATCC, HTB-44) were cultured overnight in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 1 mM non-essential amino acids, and 10% fetal bovine serum (FBS) at 37°C, 90% humidity, and 5% CO2 in either 6-well plates (for Western blotting) or 8-well slides (for confocal imaging). Cells were then treated with fresh, serum-free RPMI-1640 medium.

[0250] Cell treatment with rLon and its domain peptides

[0251] Human renal cell carcinoma cells were grown overnight in appropriate medium supplemented with fetal bovine serum (FCS) in 8-well slides (Lab-Tek II chambered no. 1.5 German cover-glass system; Thermo Fisher Scientific), 6-well plates, or 10 cm diameter culture dishes. Cells were washed and treated with 902 nM recombinant protein in serum-free medium for 3.5 hours.

[0252] The treated cells were analyzed using quantitative real-time PCR (qRT-PCR), whole-genome transcriptome analysis, confocal microscopy (63x magnification), Western blotting, or cytotoxicity assays.

[0253] Cellular uptake

[0254] Following the manufacturer's instructions (A10277, Thermo Scientific), the N-terminus of the recombinant protein was labeled with AZ647 using the NHS functional group. Free dye was removed using a dialysis chamber (D-Tube™ Dialyzer Midi), followed by centrifugation using an Amicon Ultra15 centrifuge filter (3500 xg, 40 min, 4°C). The concentration of the labeled complex was determined using a Cary UV-Vis spectrophotometer or a NanoDrop 2000c (Thermo Scientific). For live cell uptake, cells were treated with either 902 nM or 6.3 μM of recombinant protein for 3 h. Cell nuclei were counterstained with Hoechst 33342 (1:2000, molecular probe, catalog number H1399), imaged using a laser scanning confocal microscope (LSM900 confocal microscope, Carl Zeiss), and quantified using ImageJ software (NIH).

[0255] Real-time quantitative polymerase chain reaction

[0256] Total RNA was extracted from renal epithelial cells using the RNeasy Mini Kit (Qiagen) after lysis with a Qi shredder (Qiagen) in RLT buffer supplemented with β-mercaptoethanol (1%). Total RNA was reverse transcribed using the SuperScript III first-strand synthesis system (Thermo Fisher Scientific) and quantified in real-time on a RotorGene Q Cyclist (Qiagen) using the QuantiTect SYBR Green-based mRNA PCR kit (Qiagen).

[0257] Human-specific primers were used, including QuantiTect Primer Assay Hs_MYCN_1_SG (QT00201404), Hs_MYCL_1_SG (QT01004710), Hs_MYCL_va.1_SG (QT01004703), Hs_MYC_1_SG (QT00035406), Hs_IRF7_1_SG (QT00210595), and Hs_GAPDH_1_SG (QT00079247); all primers were derived from Agen. qRT-PCR was run repeatedly, and relative gene expression was calculated using the 2-ΔΔCt method.

[0258] Whole-genome transcriptome analysis of kidney cells

[0259] Total RNA was extracted from renal epithelial cells using the RNeasy Mini Kit (Qiagen) after lysis with Qiashredder (Qiagen) in RLT buffer supplemented with β-mercaptoethanol (1%). 100 ng of total RNA was precisely amplified and fragmented using Affymetrix WT PLUS reagent, and then hybridized with the Clariom S Human array (Affymetrix) at 45°C for 16 hours. The samples were then washed, stained, and scanned using the GeneChip System according to the manufacturer's instructions (Affymetrix). Data were normalized using Robust MultiAverage implemented in Transcriptome Analysis Console software (v.4.0.1.36, Applied Biosystems, Thermo Fisher Scientific). Relative expression was analyzed using empirical Bayesian methods via ANOVA; genes with an absolute fold change >1.5 were considered differentially expressed. Heatmaps were constructed in GraphPad Prism 10.

[0260] Western blot analysis

[0261] Cells were lysed using NP-40 lysis buffer (Roche Diagnostics) supplemented with protease and phosphatase inhibitors. For the preparation of nuclear extracts, cell compartments were fractionated using the Qproteome Cell Compartment kit (Qiagen). Proteins were separated by SDS-PAGE (4-12% Bis-Tris gel, Invitrogen) and imprinted onto polyvinylidene fluoride membranes (Bio-Rad), then blocked and incubated overnight at 4°C with rabbit anti-Lon (1:2000 in 5% BSA / PBS; Biorbyt, orb231326), rabbit anti-MYC (1:1000 in 5% BSA / PBS-T; Abcam, ab3968), rabbit anti-L-Myc (AvivaSystems Biology, ARP35770), or rabbit anti-NMyc (Cell Signaling Technology, 51705). The membrane was then washed, stained with goat anti-rabbit IgG horseradish peroxide-conjugated secondary antibody (1:4000 in 5% BSA / PBS-T, 1 hour at room temperature), washed again, and imaged using enhanced chemiluminescence immunoassay (GE Healthcare). Protein signals were quantified using ImageJ. β-Actin (1:4000 in PBS-T, 1 hour at room temperature; Sigma, A5316), GAPDH (1:4000 in PBS-T, 1 hour at room temperature; Santa Cruz Biotechnology, sc-25778), or histone H3 (1:4000 in PBP-T, 1 hour at room temperature; Abcam, ab1791) were used as loading controls.

[0262] Cytotoxicity test

[0263] Cells (4 × 10⁻⁶) 4 Cells (number per well) were seeded in serum-free RPMI-1640 plates (Tecan Group). Cells were treated with rLon protease or peptides of different domains and incubated for 3 hours. Cell viability or death was quantified using two different assays. Released ATP was measured using a luminescent ATP Lite kit (Perkin Elmer), and mitochondrial enzyme activity was measured using the Presto Blue assay (Invitrogen). Assays were performed in triplicate, and the average value was calculated.

[0264] In vitro degradation test

[0265] Recombinant c-MYC (CSB-EP015270HU, Cusabio) or N-MYC (AP72694, SAB Signalway antibody) protein (0.2 μg) was incubated with 0.2–1 μg rLon protease, 0.5 μg AAA+ or peptidase domain peptide (internally produced, with or without protease inhibitors), or PBS in a reaction buffer consisting of 20 mM MgCl2 at 50 mM Tris pH 8.0 in the presence of ATP (2 mM). The protein was incubated at 37 °C.

[0266] Denatured for 5 minutes at 100°C in NuPAGE LDS sample buffer (Thermo Fisher Scientific) with 10 mM dithiothreitol (DTT) for 120 minutes, followed by immunoblotting analysis using c-MYC or N-MYC antibody (Abcam, ab39688).

[0267] strain

[0268] The prototype APN strain of Escherichia coli CFT073 (O6:K2:H1) was used. 44 Incubate on trypsin-soy agar (TSA) plates at 37°C for 16 hours, harvest in phosphate-buffered saline (PBS, pH 7.2), and dilute appropriately. 18 .

[0269] To quantify the effect of Lon on bacterial survival, Escherichia coli CFT073 bacteria (CFU=10) were first tested. 4 The bacterial cultures were incubated at 37°C in 1000 μl LB medium for 3.5 hours, and then Lon (250 or 500 μg / ml) or cefotaxime (100 mg / ml) was added to the bacterial culture. OD600 (optical density at 600 nm) was measured at different time points (0, 1, 2, 3, 4 and 5 hours).

[0270] Experimental kidney infection model

[0271] C57BL / 6 background Irf3- / - The mice were provided by the RIKEN Bioresource Center in Japan, and T. Taniguchi was obtained. 45 With permission. Mice were housed at the Lund BMC animal facility at Lund University, Sweden, in individually ventilated cages (IVCs) free from specific pathogens, under a constant temperature of 23°C and a 12-hour light-dark cycle (lights were turned on at 7:00 AM), with free access to food and water. Females Irf3 / - Mice were used in the experiment at 9-15 weeks of age.

[0272] Mice were anesthetized by intraperitoneal (ip) injection of a mixture of ketamine (1.48 mg, dissolved in 100 μl of 0.9% NaCl solution, Intervet) and toluidine (0.22 mg, dissolved in 10 μl of 0.9% NaCl solution, Vetmedic), and the mixture was administered via soft polyethylene catheters (2 x 10 mm). 9 Infection was performed by intravesical inoculation of Escherichia coli CFT073 (0.05 mL containing 10 CFU / mL, 50 μl). 8 CFU) 46 Urine samples were collected from each mouse at routine time points before and after infection (24 hours, 3 days, 5 days, and 7 days) by placing the animals in sterile 1.5 ml Eppendorf tubes. Urine droplets were collected immediately, serially diluted as appropriate, and viable bacterial counts were determined by growth on TSA plates (37°C, overnight). Neutrophils in the uncentrifuged urine were counted using a hematology counter chamber. Mice were euthanized at 24 hours and 7 days after infection by anesthesia with an overdose of isoflurane (Dechra, catalog number 200-129). Organs were aseptically removed, and macroscopic pathology was documented by photographs. Tissue samples were fixed in 4% paraformaldehyde (PFA) at 4°C for 24 hours and embedded in paraffin for H&E and immunohistochemical analysis. The degree of congestion, edema, and abscess was scored on a scale of 0-10, where 0 represents similarity to the uninfected control group, and 10 represents the most severe congestion, edema, and abscess. RNA was extracted (on dry ice) and immunostained (in OCT compound or 10% (v / v) formalin) from one kidney of each mouse.

[0273] Lon protease treatment in mice

[0274] Starting the day before the bladder infection with E. coli CFT073 (day 0), administer daily... Irf3- / - Mice were intraperitoneally injected with recombinant bacterial Lon protease (rLon, 100 μl, 250 μg / ml) once for eight days. Placebo-treated mice received intraperitoneal injections of 100 μl PBS at the same schedule as Lon-treated mice. Mice were sacrificed 24 hours and 7 days later by anesthesia with an overdose of isoflurane (Dechra, catalog number 200-129).

[0275] Apc Min / + (C57BL / 6J- Apc Min / J, RRID:IMSR_JAX002020) 47,48 The mice and C57BL / 8 mice were obtained from Jackson's laboratory at approximately 8 weeks of age. Apc Min / + mice in mice Apc The (adenomatous polyposis) locus carries a heterozygous mutation, thus encoding a nonsense mutation at codon 850, leading to multiple intestinal tumors. Min C57BL / 6J- Apc Min / J mice were housed at Lund University and used at 11 weeks of age; these mice developed spontaneous intestinal adenomas. Mice were treated twice daily for 14 days by oral gavage with rLon protease (200 μl, 250 μg / ml). At sacrifice, the intestines were aseptically harvested and prepared for mRNA extraction.

[0276] Whole-genome transcriptome analysis of mouse tissues

[0277] Total RNA was extracted from mouse kidneys or intestines using the RNeasy Mini Kit (Qiagen) after lysis with the Precelys Lysis Kit (Bertin Technologies) and Tissuelyser (Qiagen) in RLT buffer supplemented with β-mercaptoethanol (1%). For treated cells, RNA was extracted using the RNeasy Mini Kit and Qiashredder (Qiagen). 100 ng of total RNA was precisely amplified and fragmented using the Affymetrix WT PLUS reagent or the GeneChip 3´IVT Express kit, and then hybridized at 45°C for 16 hours with a Clariom S Mouse HT or Mouse Genome 430 PM array (Affymetrix). The samples were then washed and stained using the Affymetrix Fluid Station 450 according to the manufacturer's instructions (hybridization, washing, and staining kit, Affymetrix).

[0278] Microarrays were scanned immediately using the GeneTitan or GeneAtlas system (Affymetrix). Data were normalized using Robust Multi-Average, implemented in Transcriptome Analysis Console software (v.4.0.1.36, Applied Biosystems, ThermoFisher Scientific). Relative expression was analyzed using Empirical Bayesian methods via ANOVA; genes with an absolute fold change ≥2.0 were considered differentially expressed. Functional analysis of differentially expressed genes was performed using Ingenuity Pathway Analysis software (IPA, Qiagen), and the data are displayed in a heatmap constructed in GraphPad Prism 9.

[0279] Immunohistochemistry

[0280] Tissues embedded and frozen in OCT were cryosectioned (8µm thick sections, Leica microtome), collected on positively charged microscope slides (Superfrost / Plus; Thermo Fisher Scientific), fixed in acetone-methanol (1:1, 10 min), infiltrated (0.2% Triton X-100, 5% normal goat serum / PBS), and incubated overnight at 4°C with rabbit monoclonal anti-c-Myc (1:100; Abcam, ab32072), rabbit anti-L-Myc (1:100, Aviva Systems Biology, ARP35770), or rabbit anti-N-Myc (1:100; Cell Signaling Technology, 51705) primary antibodies. Then, they were incubated at room temperature with Alexa 488- or Alexa 568-labeled rabbit anti-rat and goat anti-rabbit IgG secondary antibodies (1:100; molecular probes, A-21210, A-11001, and A-11011). Cell nuclei were counterstained with DAPI (4',6-diamidinyl-2-phenylindole; 0.05 mM; Sigma-Aldrich) for 15 minutes at room temperature. Slides were examined using a fluorescence microscope (AX60, Olympus Optical). Hematoxylin staining solution (Richard-Allan Scientific Signature Series Hematoxylin 7211) and Eosin-Y 7111 (Thermo Scientific) were used for counterstaining tissue sections.

[0281] Quantitative and statistical analysis

[0282] For data with a Gaussian distribution (D'Agostino and Pearson normality tests), significant differences were determined using two-tailed t-tests, one-way ANOVA with Dunn correction, or two-way ANOVA with Bonferroni multiple comparisons. For nonparametric data, two-tailed Mann-Whitney U tests or Kruskal-Wallis tests with Dunn correction were used, and paired data were analyzed using Wilcoxon paired signed-rank tests. Statistical significance was determined using GraphPad (Prism v.9), with a significance level set to [value missing]. P<0.05 P<0.01 and P < 0.001. All data are expressed as median or mean, and all error bars represent standard deviation (sd) or standard error mean (sem). Sample size (n) represents the number of independent experiments or the number of mice used in the study.

[0283] result

[0284] Identification of MYC targets of Lon and its peptides

[0285] To investigate the mechanism by which Lon inhibits MYC, as shown by Western blot analysis, the full-length Lon protein and its N-terminal domain (NTD), hexamethylene AAA+ domain, and C-terminal peptidase domain peptides were recombinantly expressed and purified via a C-terminal Strep tag. Figure 7 a, b).

[0286] Computer prediction techniques were used to identify rLon-targeted MYC protein domains and Lon peptide domains. Figure 7 ce). AlphaFold multimer scheme predictions indicate that the interaction score between full-length c-MYC and full-length Lon is low ( Figure 13 However, the resulting model revealed that the N-terminal domain of Lon is located near the C-terminal domain of c-MYC, prompting us to further analyze the interaction between these two domains. It was predicted that the Lon NTD peptide interacts with both the C-terminal portions of c-MYC (amino acid positions 356-439) and NMYC (amino acid positions 354-464), with an iPTM score of 0.697. Figure 7 c, d), but not with L-MYC (iPTM score 0.246, Figure 7 e) Interactions. A weak interaction was detected between the full-length MYC protein and the Lon peptidase domain.

[0287] Surface plasmon resonance (SPR) analysis, using Biacore technology, was employed to quantify the binding of rLon protein and Lon peptide to full-length c-MYC, NMYC, and highly conserved C-terminal domains. His-labeled full-length c-MYC or N-MYC was captured on an NTA sensor chip and then subjected to dose-dependent binding analysis with rLon protein or Lon peptide. rLon and NTD peptides bound to His-labeled c-MYC and N-MYC in a dose-dependent manner. D In the range of approximately 30 to 40 μM. Furthermore, the NTD peptide binds to the His-tagged C-terminal domain of c-MYC ( Figure 7f). SPR analysis further detected the binding of the peptidase and AAA+ domains to the full-length c-MYC. The binding affinity of rLon and its domains is similar to that of MYCMI-6, a small molecule inhibitor of Myc-driven transcription, which binds to the MYC bHLHZip domain. 23 The significant difference between the lack of computer-predicted peptidase domains and SPR-positive analysis confirms that AlphaFold cannot clearly provide folding patterns within intrinsically disordered regions that are often targets of degradation. 24 Due to the lack of the full-length L-MYC protein, the binding interaction between the full-length Lon and Lon peptide and L-MYC could not be determined by SPR analysis.

[0288] The structural superposition of the crystal structure with that of the MYC-MAX complex shows that the NTD peptide almost completely overlaps with the MAX bound to MYC. Figure 7 (g) indicates that the NTD targets the MAX binding site, which is the mechanism by which Lon substitutes for MAX and reduces the formation of the MYC-MAX complex. This hypothesis was addressed using the ELISA-based TransAM c-MYC assay, which quantifies the binding of c-MYC to its shared E-box DNA sequence. The rLon protein and the NTD and AAA+ domains reduced the binding of c-MYC to DNA ( Figure 7 h). The function of the NTD domain is consistent with computer predictions of the NTD binding MAX binding site, and the function of the AAA peptide is consistent with the known affinity of this domain for DNA. 25 .

[0289] Cellular uptake of Lon and Lon peptide domains and their effects on cellular MYC levels

[0290] Lon proteases have been shown to enter human cells and reduce intracellular MYC levels, but the cell-cell interactions of Lon peptides have not been previously investigated. To address this issue, human renal cell carcinoma cells were treated with each Lon peptide, and visualization and quantification were performed using AZ647 fluorescent labeling. Each AZ647-labeled peptide exhibited a similar cellular uptake pattern. Figure 8 a) There was obvious staining in the cytoplasm and nucleus, which was also confirmed by Western blot analysis of whole-cell extracts from cells exposed to rLon or each peptide. Figure 8 b; Figure 9 The complex pattern indicates that rLon fragmentation generates peptide domains.

[0291] To quantify the overall effect on cellular c-MYC, N-MYC, and L-MYC protein levels, human kidney cells treated with rLon or each Lon peptide were fixed and imaged by confocal microscopy after staining with anti-c-MYC-, N-MYC-, or L-MYC-specific antibodies. Compared to control cells, c-MYC, N-MYC, and L-MYC showed similar patterns of reduced staining after rLon and NTD treatment. Figure 14 In contrast, cells treated with peptidase domains did not show a significant decrease in c-MYC or N-MYC signaling, but intracellular redistribution from the cytoplasm to the nucleus was detected. Cytoplasmic c-MYC signaling was significantly attenuated, while nuclear and perinuclear c-MYC signaling was enhanced, indicating increased c-MYC translocation and / or activation. Figure 8 c, d). Furthermore, rLon and NTD peptides significantly reduced L-MYC levels. However, unlike other MYC members, L-MYC signaling was also reduced in peptidase-treated cells compared to the control group ( Figure 8 e). AAA+ domain peptides decreased N-MYC levels but increased c-MYC and L-MYC levels.

[0292] The results confirmed that rLon treatment reduced cellular MYC levels and identified NTD as a potent inhibitor of C-, N-, and L-MYC in therapeutic cells.

[0293] Mechanism by which Lon and Lon domain peptides inhibit MYC gene expression

[0294] The inhibition of MYC / MAX complex formation by Lon and NTD peptides suggests that gene expression is affected in cells treated with rLon or NTD. Therefore, using MYC , MYCN or MYCL (Isomer I and II) specific primers, quantified by qRT-PCR MYC Express( Figure 8 f). Compared with PBS-treated control cells, rLon or Lon domain peptides showed better efficacy. MYC Expression was not significantly regulated (FC<2). MYCN The expression regulation was weaker (FC-2.1 and -2.7). In contrast, human kidney cells exposed to rLon (FC-9.7) showed significantly higher expression levels compared to control cells. MYCL The expression of the peptide was strongly suppressed, and the inhibition was further enhanced in cells exposed to the NTD peptide (FC-38.4) or the peptidase domain peptide (FC-21) compared with the control.

[0295] The effects of Lon on MYC-dependent gene expression were further investigated in cells exposed to rLon, NTD, peptidase, or AAA+ peptide. Genome-wide transcriptome analysis identified MYC targets that were significantly inhibited in human renal cell carcinoma cells treated with NTD and AAA+ peptide, while the effects of rLon and peptidase domains were weaker, consistent with in vitro data on MYC-MAX inhibition. Figure 8 (g, h). Repressed genes include zinc finger proteins (ZFPs), suggesting a mechanism that influences transcriptional mechanisms.

[0296] Mechanism of Lon and Lon peptide domain degradation of MYC

[0297] Through ATP and Mg 2+ rLon was mixed with recombinant c-MYC or N-MYC in the presence of PBS, and the direct degradation of MYC by Lon was quantified in vitro. Compared with PBS, after incubation with rLon at 37°C for 120 minutes, Coomassie staining and Western blot analysis showed a significant decrease in c-MYC protein levels. Figure 9 a) Using the same protocol, the Lon peptidase domain significantly reduced c-MYC and N-MYC protein levels, and this effect was enhanced in the presence of the ATP-binding AAA+ domain, which is essential for complete peptidase activity in bacteria. 26 ( Figure 9 b). In the presence of protease inhibitors, degradation is inactivated ( Figure 9 c). Full-length L-MYC cannot be used for in vitro analysis.

[0298] To quantify the effect of rLon treatment on cellular MYC protein levels, Western blot analysis was performed on whole-cell extracts. Figure 9 df). In cells exposed to full-length rLon, c-MYC protein levels were significantly reduced, with this effect being particularly pronounced in nuclear extracts compared to cells treated with PBS. However, c-MYC levels were not affected by individual peptide domains ( Figure 9 df). Cells treated with full-length rLon or peptidase domains from whole-cell lysates and nuclear extracts showed significantly reduced N-MYC protein levels after rLon treatment. Figure 9 df). Compared with c-MYC and N-MYC, rLon treatment significantly reduced L-MYC levels, including isoform II (20 kDa) and isoform I (40 kDa), and was strongly influenced by NTD, AAA+, and peptidase domains, although these dissociated domains primarily affected the shorter isoform (df). Figure 9 df).

[0299] The results indicate that the MYC transcription factor family is a target of bacterial Lon proteases, which are taken up by human cells and affect cellular MYC levels in multiple ways. First, computational analysis predicted that the MAX binding site is a target of the Lon NTD domain, which was shown to inhibit MYC-MAX heterodimerization, crucial for DNA binding to the helical-loop-helical domain that subsequently initiates MYC-dependent transcription. 27 Secondly, in vitro studies demonstrated that MYC degradation is jointly carried out by the peptidase domain and the ATP-binding AAA+ domain, suggesting that the decrease in c-MYC, N-MYC, and L-MYC levels after rLon treatment can be attributed to these two mechanisms. Interestingly, the regulation of L-MYC is attributed to transcriptional effects and the resulting loss of c-MYC and N-MYC activation of L-MYC expression, rather than the degradation of the peptidase and AAA+ domain.

[0300] Lon treatment can suppress the MYC response to infection in susceptible disease mice.

[0301] carry Irf3 Mice lacking this feature are more susceptible to kidney infection. Irf3- / - Mice are models of human diseases. 28,29 Therefore, in Irf3- / - The response of MYC to kidney infection was studied in mice infected with urinary tract pathogenic Escherichia coli strain CFT073 via intravesical injection. Figure 10 a). Whole-genome transcriptome analysis was performed on total renal RNA collected from infected or uninfected mice, and disease-related genes were identified by comparing infected mice with uninfected controls at 24 hours and 7 days later.

[0302] Myc The expression of Myc-dependent genes increased after infection (compared to uninfected controls, 486 genes significantly regulated MYC-related genes at 24 hours and 786 genes significantly regulated MYC-related genes at 7 days), and most of these genes were activated. Figure 4 (b, c). Compared with the uninfected control group, the increase in total gene expression showed a kinetic similar to that of MYC-dependent genes. Further renal tissue analysis showed that, compared with the uninfected control group, the increase in total gene expression... Irf3- / - Elevated c-Myc protein levels in the kidneys of mice, particularly along the inner lining of the renal pelvis and the renal papillae, which are the primary sites of infection. Figure 10 e, f).

[0303] Next, we investigated whether Lon acts as a MYC inhibitor in this model. Irf3- / - Mice were injected intraperitoneally with recombinant Lon protease (rLon, 250 μg / ml). -1Once daily for eight days, starting the day before E. coli CFT073 bladder infection. Figure 10 a). Kidney was detected in the rLon treatment group. Myc Gene expression was significantly reduced (on day 7, compared to 786 genes in the placebo group, 2 genes were significantly regulated). Figure 10 c, d). Kidney tissue analysis showed that, compared with the placebo group, rLon treatment reduced infection rates. Irf3- / - The reduction in c-Myc staining in mice confirmed the therapeutic effect at the protein level. Figure 10 e, f).

[0304] Functional analysis of MYC-dependent genes showed that innate immunity was the most strongly regulated gene class in the placebo group. Figure 4 g), c-MYC-dependent innate immune regulatory factors are activated; Toll-like receptors, IL-1β, tumor necrosis factor (TNF), type I interferon, and calcium-binding proteins (S100A8, S100A9) that activate neutrophil recruitment are activated. Figure 4 g). rLon treatment almost eliminated the MYC-dependent innate immune response ( Figure 4 h). In rLon-treated mice, major innate immune signaling pathways were no longer regulated, including interferon, Toll-like receptors, NF-κB, and inflammasome signaling pathways (h). Figure 10 i). The results showed that, compared with the placebo group, infection triggered rLon treatment. Irf3- / - The c-MYC dysregulation and excessive innate immune response in mice were suppressed or almost eliminated.

[0305] rLon treatment suppresses the activation of L-MYC and N-MYC in infection and cancer models.

[0306] To investigate whether the effects of rLon extend to other MYC family members, specific analyses of L-MYC and N-MYC-dependent gene expression were performed on the RNA dataset. Figure 11 (ad). Compared with uninfected control mice, the expression of L-Myc and N-Myc was significantly increased at 24 hours and 7 days post-infection. Approximately 25% of the genes were annotated as associated with N-Myc, and 33% as associated with L-Myc, showing increased expression. Further increases in L-Myc and N-Myc staining along the renal pelvis and papillae confirmed infection. Irf3- / - N-Myc and L-Myc responses at the protein level in mice (7 days, Figure 11 e, f).

[0307] At 24-hour and 7-day time points, Irf3- / -rLon treatment in mice almost eliminated the response of these L-Myc and N-Myc to infection. Figure 11 af). Seven days post-infection, rLon-treated mice showed no L-MYC-dependent genes, only differential expression of two N-MYC-related genes ( Figure 11 b, d), infected with rLon treatment Irf3- / - L-MYC and N-MYC staining in mouse tissues was suppressed ( Figure 5 e, f). These results indicate that in Irf3- / - In mice, N-Myc and L-Myc are activated by infection, and rLon inhibits the dysregulation of N-Myc and L-Myc in infected kidneys.

[0308] To address whether the effects of rLon on L-MYC and N-MYC expression would translate into other disease states, the rLon-treated group was examined compared to the placebo group. Apc Min / + A mouse RNA dataset was used to understand changes in N-MYC and L-MYC-dependent gene expression. Figure 11 gm). In the placebo group, tumor-bearing Apc Min / + N-Myc and L-Myc expression in mice was significantly activated ( Figure 11 hj), but not in the rLon processing group ( Figure 11 These results indicate that N-Myc and L-Myc in km). Apc Min / + The cytokines were deregulated in the mouse gut, and rLon acted as a pan-MYC inhibitor in these bacterial infection and cancer models.

[0309] RLon-treated mice were protected from nephrotoxicity.

[0310] Acute pyelonephritis is a major cause of acute kidney tissue destruction and persistent kidney damage. 30 Therefore, we investigated whether genes regulating nephrotoxicity were affected by rLon treatment. Compared to the uninfected control group, a large number of kidney injury-related genes were regulated in the placebo group, of which 93 / 271 genes were defined as MYC-related (34%). Figure 12 a, b). In contrast, in the rLon untreated group, only one gene associated with nephrotoxicity was significantly regulated, and there was no evidence of a broader MYC-dependent renal injury response. Figure 12 The results showed that rLon targets and inhibits genes associated with nephrotoxicity, kidney injury, or kidney-specific cell death, which can lead to kidney damage.

[0311] These results suggest that Lon may target disease-associated MYC dysregulation more strongly than physiological MYC expression and tissue homeostasis. This can be achieved by quantifying healthy, uninfected individuals. Irf3- / - Infections in mice treated with rLon or placebo Irf3 / - The study addressed this issue by investigating homeostatic MYC-dependent gene expression in mice. rLon treatment had no significant effect on disease-independent gene expression, and treated mice remained similar to uninfected mice. Principal component analysis of the RNA dataset showed that the rLon-treated group clustered adjacently with uninfected mice, while the placebo group clustered significantly differently from healthy mice (90% variance, first principal component). Figure 12 e).

[0312] Further construct volcano maps to visualize uninfected individuals. Irf3- / - Overall differences in gene expression among mice, rLon-treated mice, and placebo-treated mice, including genes expressed but not differentially regulated by infection, and the effect of Lon treatment on these gene classes. Figure 12 (fh). The disease response comprises 3695 genes, including 786 MYC-related genes, which are associated with uninfected individuals. Irf3- / - Compared to mice, the placebo group showed differences in gene expression. Figure 12 f, g). In contrast, compared to uninfected Irf3- / - Compared to mice, these genes showed little differential expression in untreated rLon mice (n=43). Figure 12 (f, g), indicating that rLon targets the disease-related increase in gene expression that occurred in the placebo group.

[0313] These results indicate that Lon primarily suppresses infection-induced MYC dysregulation without significantly disrupting healthy MYC gene expression mechanisms.

[0314] discuss

[0315] This study explored the mechanism by which Lon inhibits MYC and identified two parallel mechanisms of action: one is the NTD blocking of the formation of the c-MYC and N-MYC-MAX complex, thereby interfering with MYC-driven gene expression; the other is the degradation of MYC protein by a peptidase domain enhanced by the AAA+ domain. No off-target effects were detected, indicating that rLon treatment primarily affects MYC dysregulation in these disease models. These results collectively demonstrate that Lon proteases are a novel, multifunctional tool for neutralizing the disruptive effects of MYC dysregulation in various disease states.

[0316] Within the MYC family, c-MYC is the most studied. This short-lived protein is degraded via the ubiquitin-proteasome pathway, depending on the phosphorylation state of Thr58 and Ser-62, with phosphorylated Thr-58 being recognized by the Fbxw7 E3 ubiquitin ligase. 31,32 Conversely, ubiquitin-specific proteases can deubiquitinate and protect MYC by counteracting Fbw7alpha and Fbw7gamma. 33,34 MYC phosphorylation, ubiquitination, and proteasome degradation have become key mechanisms for controlling MYC levels in cancer cells. 7，14 The research targets a wide range of compounds, several of which are currently undergoing preclinical development or early clinical trials. 7，35 These include all-trans retinoic acid (ATRA) and Vismodegib, which are FDA-approved for the treatment of acute myeloid leukemia and basal cell carcinoma. 36，37 In our study, with Omomyc 38,39 Conversely, the effect of rLon was independent of the phosphorylation status of Thr-58 and Ser-62 in the cellular model. An intriguing explanation for the Lon effect is that bacterial Lon peptidases possess an inherent function as unfolded protein scavengers, capable of degrading disordered MYC domains, thereby bypassing specific mammalian phosphorylation checkpoints that would otherwise protect cancer-dysregulated MYC. 14,40,41 Our computer data predict that the additional effect of Lon NTD in inhibiting MYC-MAX will further reduce MYC expression levels, especially L-MYC, in cells and tissues.

[0317] Inhibitors targeting dysregulated MYC ideally affect most human cancers, but selective inhibitors are difficult to find, and the off-target effects of MYC inhibitors are a concern. This study demonstrates that bacterial Lon protease effectively enhances antibacterial defense against kidney infection and prevents histopathology by preventing dysregulation of MYC in infected tissues. Notably, rLon treatment in infected mice resulted in near-complete shutdown of nephrotoxic genes. Lon also affected the levels of c-MYC, N-MYC, and L-MYC proteins in infected and tumor-bearing mice.

[0318] These potent effects are significant, especially since there is no evidence that rLon targets normal MYC expression. The molecular basis for this apparent specificity is unclear, but several possibilities can be proposed. First, Lon proteases may target disease-specific modifications of MYC, optimizing Lon binding and the degradation of its targets. 14,40,41 Secondly, MYC overexpression may make the protease more likely to perform its proteolytic function. 41Third, in cancer or infection, Lon access to the MYC promoter, particularly the NTD and AAA+ domains, may be enhanced in the open chromatin of rapidly dividing cells. This could explain the significant transcriptional repression detected in diseased tissues but not in healthy tissues. 42 .

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Oncogenic activity of the c-Myc protein requiresdimerization with Max. Cell 72, 233-245 (1993). 28 Fischer, H. et al. Pathogen specific, IRF3-dependent signaling andinnate resistance to human kidney infection. PLoS Pathog. 6, e1001109 (2010). 29 Puthia, M. et al. IRF7 inhibition prevents destructive innateimmunity—A target for nonantibiotic therapy of bacterial infections. Sci. Transl. Med. 8, 336ra359336ra359 (2016). 30 Roberts, J. A. Management of pyelonephritis and upper urinarytract infections. Urol. Clin. North Am. 26, 753-763 (1999). 31 Yada, M. et al. Phosphorylation dependent degradation of c Myc ismediated by the F box protein Fbw7. EMBO J. 23, 2116-2125 (2004). 32 Welcker, M. et al. The Fbw7 tumor suppressor regulates glycogensynthase kinase 3 phosphorylation-dependent c-Myc protein degradation. Proc. Natl. Acad. Sci. U. S. A. 101, 9085-9090 (2004). 33 Popov, N. et al. The ubiquitin-specific protease USP28 is requiredfor MYC stability. Nat. Cell. Biol. 9, 765-774 (2007). 34 Sun, X. X. et al. The nucleolar ubiquitin-specific proteaseUSP36deubiquitinates and stabilizes c-Myc. Proc. Natl. Acad. Sci. U. S. A. 112,37343739 (2015). 35 Sun, X. X., Li, Y., Sears, R. C.&Dai, M. S. Targeting the MYCubiquitinationproteasome degradation pathway for cancer therapy. FrLont. Oncol. 11, 679445 (2021). 36 Giuli, M. V. et al. Current trends in ATRA delivery for cancertherapy. Pharmaceutics 12, 707 (2020). 37 Axelson, M. et al. US Food and Drug Administration approval:vismodegib for recurrent, locally advanced, or metastatic basal cellcarcinoma. Clin. Cancer Res. 19, 2289-2293 (2013). 38 Truica, M. I., Burns, M. C., Han, H.&Abdulkadir, S. A. Turning upthe heat on MYC: Progress in small-molecule inhibitors. Cancer Res. 81, 248-253(2021). 39 Han, H. et al. Small-molecule MYC inhibitors suppress tumor growthand enhance immunotherapy. Cancer Cell 36, 483-497.e415 (2019). 40 Madden, S. K., de Araujo, A. D., Gerhardt, M., Fairlie, D. P.&Mason, J. M. Taking the Myc out of cancer: toward therapeutic strategies todirectly inhibit cMyc. Mol. Cancer 20, 3 (2021). 41 Wolf, E.&Eilers, M. Targeting MYC proteins for tumor therapy. Annu. Rev. Cancer Biol. 4, 61-75 (2020). 42 Arends, J. et al. An integrated proteomic approach uncovers novelsubstrates and functions of the Lon protease in Escherichia coli . Proteomics 18,1800080 (2018). 43 Mirdita, M. et al. ColabFold: making protein folding accessible toall. Nat. Methods. 19, 679-682 (2022). 44 Mobley, H. et al. Pyelonephritogenic Escherichia coli and killing ofcultured human renal proximal tubular epithelial cells: role of hemolysin insome strains. Infect. Immun. 58, 1281-1289 (1990). 45 Sato, M. et al. Distinct and essential roles of transcriptionfactors IRF-3 and IRF-7 in response to viruses for IFN-α / β geneinduction. Immunity 13, 539-548 (2000). 46 Edén, C. S. et al. Inhibition of experimental ascending urinarytract infection by an epithelial cell-surface receptor analogue. Nature 298,560-562 (1982). 47 Moser, A. R., Pitot, H. C.&Dove, W. F. A dominant mutation thatpredisposes to multiple intestinal neoplasia in the mouse. Science 247, 322-324(1990). 48 Su, L.-K. et al. Multiple intestinal neoplasia caused by a mutationin the murine homolog of the APC gene. Science 256, 668-670 (1992).

Claims

1. A peptide comprising an amino acid sequence substantially identical to one or more domains of a Lon protease or a functional fragment or variant thereof.

2. The peptide according to claim 1, wherein the Lon protease domain is an N-terminal domain.

3. The peptide of claim 2, wherein the N-terminal domain comprises part or all of the α-helix region.

4. The peptide of claim 2, wherein the N-terminal domain has partially or completely deleted from the α-helix region.

5. The peptide according to claim 1, wherein the Lon protease domain is an AAA+ domain.

6. The peptide according to claim 1, wherein the Lon protease domain is a C-terminal proteolytic domain.

7. The peptide according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence substantially identical to the amino acid sequence of the first Lon protease domain and an amino acid sequence substantially identical to the amino acid sequence of the second Lon protease domain.

8. The peptide according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence substantially identical to only one or only two domains of a Lon protease.

9. The peptide according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence substantially identical to the amino acid sequence of the N-terminal domain of a Lon protease and an amino acid sequence substantially identical to the amino sequence of another domain.

10. The peptide according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence substantially identical to the amino acid sequence of the C-terminal proteolytic domain of a Lon protease and an amino acid sequence substantially identical to the amino acid sequence of another domain.

11. The peptide according to any one of the preceding claims, wherein the peptide comprises an amino acid sequence substantially identical to only one or only two domains of a Lon protease.

12. The peptide according to any one of the preceding claims, wherein the peptide does not contain the amino acid sequence of the intact wild-type Lon protease.

13. The peptide according to any one of the preceding claims, wherein the Lon protease is of bacterial origin.

14. The peptide according to any one of the preceding claims, wherein the length of the peptide is less than about 780 amino acids.

15. The peptide according to any one of the preceding claims, wherein the peptide is longer than about 110 amino acids.

16. The peptide according to any one of claims 1 to 4 and 7 to 15, comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID No. 2 or 3 or a fragment or variant thereof.

17. The peptide according to any one of claims 1, 5 and 7 to 15, comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID No. 4 or a fragment or variant thereof.

18. The peptide according to any one of claims 1 to 15, comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID No. 5 or a fragment or variant thereof.

19. Peptides, variants, or fragments may be isolated, substantially isolated, purified, or substantially purified. If a peptide, variant, or fragment contains no other components, such as lipids or other peptides, it is isolated or purified. If a peptide, variant, or fragment is mixed with a carrier or diluent that does not interfere with its intended use, it is substantially isolated or substantially purified. For example, if a peptide, variant, or fragment exists in a form containing less than 10%, less than 5%, less than 2%, or less than 1% of other components, it is substantially isolated or substantially purified.

20. A polynucleotide encoding a peptide according to any of the preceding claims.

21. A vector comprising the polynucleotide of claim 20.

22. A host cell comprising the polynucleotide of claim 20 or the vector of claim 21.

23. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 19, a polynucleotide according to claim 20, or a carrier according to claim 21 and a pharmaceutically acceptable carrier.

24. The peptide according to any one of claims 1 to 19, the polynucleotide according to claim 20, the carrier according to claim 21, or the pharmaceutical composition according to claim 23 is used for treatment.

25. The peptide according to any one of claims 1 to 19, the polynucleotide according to claim 20, the carrier according to claim 21, or the pharmaceutical composition according to claim 23, for the treatment or prevention of infection, particularly bacterial infection, or for the treatment of cancer, or for the treatment of diseases regulated by L-MYC, N-MYC, or C-MYC, or for the regulation of L-MYC, N-MYC, and C-MYC.

26. A method for treating or preventing infection or cancer, or treating a disease regulated by L-MYC, N-MYC, or C-MYC, or regulating L-MYC, comprising the step of administering an effective amount of the peptide according to any one of claims 1 to 19, the polynucleotide according to claim 20, the carrier according to claim 21, or the pharmaceutical composition according to claim 23 to a subject in need.

27. A peptide comprising an amino acid sequence having an amino acid sequence of a Lon protease or a functional variant or fragment thereof, for treating diseases such as infection or cancer, wherein the disease is regulated by L-MYC or N-MYC.

Images