Peptide probes

Abstract

This invention provides peptide probes according to formula (I) as defined herein. The peptide probes of this invention have been found to be useful for detecting, screening, monitoring, classifying, selecting treatments, determining the effectiveness of treatments, and / or predicting urinary tract infections. This invention also provides compositions comprising the peptide probes of this invention, methods for detecting and treating urinary tract infections using said peptide probes, and in vitro diagnostic kits comprising the peptide probes of this invention.

Description

Technical Field

[0001] This invention relates to peptide probes that can be used to detect, screen, monitor, classify, select treatments for urinary tract infections, determine the effectiveness of treatment, and / or predict urinary tract infections. The invention also provides compositions comprising the peptide probes of this invention, methods for detecting and / or treating urinary tract infections using said peptide probes, and in vitro diagnostic kits comprising the peptide probes of this invention. Background Technology

[0002] The listing or discussion of previously disclosed documents in this specification should not necessarily be construed as an admission that such documents are part of the prior art or common knowledge.

[0003] Escherichia coli ( E. coli Foodborne illness (FIO) is one of the most common foodborne and waterborne pathogens in the world, infecting millions and causing adverse conditions such as urinary tract infections, diarrhea, bloodstream infections, and respiratory illnesses (WHO, WHO Assessment of the Global Burden of Foodborne Diseases, World Health Organization, 2015). However, despite the urgent need for detection, the standard method currently used for detection remains routine bacterial isolation and culture, which can be extremely time-consuming and labor-intensive (Wang and Salazar, Comprehensive Reviews in Food Science and Food Safety 2016, 15, 183-205 and Lazcka). et al (Biosens Bioelectron 2007, 22, 1205-1217). In cases of persistent infection, antibiotics are prescribed to alleviate the symptoms, but this inadvertently leads to an undesirable surge in antibiotic resistance (Bartoletti). et al [References: ., European Urology Supplements 2016, 15, 81-87, and EAU, EAU Guidelines on Urological Infections, European Association of Urology, 2022]. Due to the increasing annual mortality rate from ingestion of food and water contaminated with E. coli, several research groups have recently reported functional assays for the rapid detection of the presence of E. coli in food and water samples. However, these methods remain primarily limited to in vitro diagnostic kits, which greatly facilitate pre-detection to prevent subsequent ingestion of contaminated substances (Wang and Salazar, Comprehensive Reviews in Food Science and Food Safety 2016, 15, 183-205, Law). et al., Front Microbiol 2014, 5, 770, and Sinsinbar et al (AngewChem Int Ed Engl 2020, 59, 18068-18077). However, rather than simply adopting preventative strategies, it is more important to fully investigate and understand the underlying biological mechanisms associated with the global mortality rate of E. coli infection. Unfortunately, such research remains elusive to date.

[0004] As part of the symbiotic gut microbiota, it is not surprising that *Escherichia coli* is generally lacking in virulence in the human gastrointestinal tract. Even so, they can still cause intestinal disease after ingestion of pathogenic strains with significant virulence and an innate ability to overcome host defenses (Mueller). et al ., Treasure Island (FL): StatPearlsPublishing, 2023). However, all these effects can be greatly mitigated with highly regulated safety standards. On the other hand, extraintestinal diseases caused by E. coli are usually caused by the translocation of symbiotic intestinal bacteria to other parts of the body, resulting in life-threatening conditions such as pneumonia (lung), bacteremia (blood), and meningitis (brain) (Mueller). et al ., Treasure Island (FL): StatPearls Publishing, 2023, Mylotte et al .,Clinical Infectious Diseases 2002, 35, 1484-1490, McCue, Journal of the American Geriatrics Society 1987, 35, 213-218, and Jain et al (N Engl J Med 2015, 373, 415-427). The urethra, in particular, is the most common site of extraintestinal infection caused by *E. coli* ascending from the intestines via trace amounts of fecal matter (Zagaglia). et al (., Microorganisms 2022, 10). Therefore, urinary tract infections (UTIs) are among the most common diseases encountered in global clinical practice, affecting 150 million people annually, with a considerably high incidence and high medical costs (Flores-Mireles). et al ., Nat Rev Microbiol 2015, 13, 269-284, and Sihra et al(Nat Rev Urol 2018, 15, 750-776). A surge in resistance rates to antibiotics recommended in current UTI treatment guidelines and an increase in the number of multidrug-resistant urinary tract pathogenic Escherichia coli (UPEC) isolates were observed (Bruxvoort, 2018, 15, 750-776). et al ., Clin Infect Dis 2020, 71, 100-108, and Mazzariol et al (J Chemother 2017, 29, 2-9), thus prompting not only an urgent need for new treatments, but also a deeper understanding of biology to improve the current situation.

[0005] Routine methods for identifying bacteria include culturing and colony counting, which can be labor-intensive, time-consuming, and often require a series of complex analyses before reliable results can be obtained (Velusamy). et al (Biotechnol Adv 2010, 28, 232-254). Therefore, these conventional methods limit their application in bacterial detection. Over the years, various techniques have emerged to overcome these limitations in bacterial detection, such as polymerase chain reaction (PCR). et al ., Biosens Bioelectron 2007, 22, 1205-1217), electrochemical sensing (Su et al ., Biosens Bioelectron 2011, 26, 1788-1799), Immunological methods (Swaminathan) et al ., Annual Reviews Microbiology 1994, 48, 401-426), mass spectrometry and chromatography (Zhu et al (Chem Sci 2016, 7, 2987-2995). However, these methods require sophisticated equipment and involve complex procedures. Furthermore, in clinical practice, traditional methods for detecting bacterial infections rely primarily on blood analyses, such as the procalcitonin test (PCT) and C-reactive protein (CRP); and / or nuclear imaging, such as magnetic resonance imaging (MRI), computed tomography (CT), X-ray, and positron emission tomography (PET) (Boyles and Wasserman, South African Medical Journal 2015, 105, and Polvoy). et al(J Nucl Med 2020, 61, 1708-1716). However, these methods are invasive and inefficient, and may provide false positive results, which will delay the entire diagnostic process (Yoon). et al ., Chem2021, 9, 743923).

[0006] Therefore, there is a need for improved or alternative methods to detect bacteria, particularly urinary tract pathogenic Escherichia coli, preferably methods with high specificity and sensitivity that do not require complex machinery and invasive procedures. Summary of the Invention

[0007] This invention provides a peptide probe according to formula (I):

[0008] in, P does not exist or is -C(O)C 1-6 alkyl; Z represents a peptide containing the amino acid sequence according to formula (II):

[0009] in, n is an integer from 0 to 6; m is an integer from 0 to 5; Each X 1 Independently selected from any amino acid; X 3 For Arg or Lys; Each X 4 Independently selected from any amino acid; When m is 0, X 5 It does not exist or is selected from any amino acid; and When m is between 1 and 5, X 5 Selected from any amino acid; L represents a bond or a self-splitting linker; and Q is a detectable marker; or The peptide probe salt or solvate.

[0010] In some preferred embodiments, in formula (II), when each X 1 X 4 and X 5 When present, they are independently selected from any standard amino acid or its D-amino acid. For example, in formula (II), each X 1 It can be independently selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; each X 4It can be independently selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; and when m is 0, X 5 It may be absent or selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; and when m is 1 to 5, X 5 It can be selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro or their D-amino acids.

[0011] The present invention also provides compositions comprising the peptide probe and carrier of the present invention, wherein in some embodiments, the compositions are pharmaceutical compositions comprising the peptide probe of the present invention and a pharmaceutically acceptable carrier.

[0012] This invention also provides a method for detecting urinary tract infection in a urine sample obtained from a subject, and a method for determining one or more suitable therapeutic agents for treating urinary tract infection in a subject. This document also discloses methods for determining one or more suitable therapeutic agents for treating urinary tract infection in a subject, and methods for treating urinary tract infection in a subject. This document further provides in vitro diagnostic kits for detecting, screening, monitoring, classifying, selecting treatment for urinary tract infection in a subject, determining whether treatment is effective in a subject's urinary tract infection, and / or predicting urinary tract infection in a subject, said in vitro diagnostic kits comprising one or more peptide probes of this invention.

[0013] Preferred but optional features are set forth in the dependent claims. Other aspects and embodiments of the peptide probes, compositions, methods, and kits of the present invention will be apparent from the following description, drawings, and claims. It will be understood from the above and below description that each feature described herein, as well as each combination of two or more such features, is included within the scope of this disclosure, provided that the features included in such combinations do not contradict each other. Furthermore, any feature or combination of features may be expressly excluded from any embodiment. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the synergistic activation of NO-AH (compound 7) by OmpT and aminopeptidase N (APN) in the presence of urinary tract bacterial infection.

[0015] Figure 2 The synthesis schemes for R-CyOH are shown: (a) K2CO3, CH3CN, 50℃, 6 hours; (b) N(c) 1. PBr3, anhydrous tetrahydrofuran (THF), 0 °C, 2 h; 2. N,N-diisopropylethylamine (DIPEA), 55 °C, 6 h; (d) 1. DCM, triisopropylsilane (TIPS), trifluoroacetic acid (TFA), 0 °C; 2. 5% piperidine in dimethylformamide (DMF) solution, room temperature.

[0016] Figure 3 The synthesis schemes for NO-AH are shown as follows: (a) K2CO3, CH3CN, 50 °C, 6 h; (b) EEDQ, anhydrous DCM, room temperature, 16 h; (c) 1. PBr3, anhydrous THF, 0 °C, 2 h; 2. K2CO3, 50 °C, 4 h; (d) DCM, TIPS, TFA, 0 °C.

[0017] Figure 4 The in vitro stability of NO-AH in phosphate-buffered saline (PBS) is shown. (a) Absorbance of NO-AH (10 μM) at 660 nm after incubation in PBS at 37 °C for 0, 2, 18, and 24 hours. (b) Fluorescence emission of NO-AH (10 μM) at 720 nm after incubation in PBS at 37 °C for 0, 2, 18, and 24 hours. Data are expressed as mean ± SD (n = 3).

[0018] Figure 5 shows the in vitro characterization of NO-AH. (a) Schematic diagram of NO-AH digestion by OmpT and APN. OmpT digestion of the peptide probe resulted in the release of a peptide with the sequence RFFR (SEQ ID NO: 5). (bc) Absorbance and fluorescence spectra of NO-AH (10 μM) in PBS (pH 7.4) at 37 °C for 2 hours in the presence and absence of both OmpT and APN. (λex: 660 nm) (d) HPLC analysis of OmpT and APN after co-incubation with NO-AH in PBS (pH 7.4) at 37 °C for 2 hours. (e) Enzyme kinetics of OmpT with NO-AH (10 μM). (f) Enzyme kinetics of APN with R-CyOH (10 μM). (g) Selectivity analysis of the enzymatic NO-AH reaction (10 μM) after co-incubation with the specified biomolecule in PBS at 37 °C for 2 hours. (λex: 660 nm; λem: 720 nm). (F represents the fluorescence emission of the specified biomolecule at λem: 720 nm; F0 represents the baseline fluorescence of NO-AH at λem: 720 nm). Data are expressed as mean ± SD (n = 3).

[0019] Figure 6 The following are shown: (a) absorption spectrum and (b) fluorescence spectrum of NO-AH (10 μM) incubated in PBS (pH 7.4) at 37 °C for 2 h in the presence of a single enzyme (OmpT or APN) and both enzymes.

[0020] Figure 7 The HPLC chromatograms of a single enzyme (OmpT or APN) are shown. The enzyme was incubated with NO-AH (10 μM) in PBS (pH 7.4) at 37°C for 2 hours.

[0021] Figure 8 The following HPLC spectra are shown: (a) Time-dependent HPLC spectra of R-CyOH formation at different time points in PBS (pH 7.4) at 37 °C, when OmpT protein hydrolyzes NO-AH (10 μM). (b) Time-dependent HPLC spectra of CyOH formation at different time points in PBS (pH 7.4) at 37 °C, when OmpT protein hydrolyzes R-CyOH (10 μM).

[0022] Figure 9 The in vitro fluorescent bacteria (approximately 10 μM) of different bacterial strains after co-incubation with NO-AH (10 μM) in the presence and absence of OmpT or APN inhibitors are shown. 8 Confocal microscopy analysis (CFU / mL). (ab) Confocal images of different bacterial strains co-incubated with NO-AH (10 μM) in PBS at 37°C for 2 hours, including CFT073, UTI89, J96, MRSA, and Enterococcus faecalis (CFU / mL). E. faecalis ) and Proteus mirabilis ( P. mirabilis (λex: 640 nm, λem: 700 / 30 nm) Scale bar: 2.5 μm. (ce) Confocal images of urethropathogenic Escherichia coli bacteria co-incubated with NO-AH (10 μM) in the presence and absence of OmpT inhibitor (Ac-RffRr) and APN inhibitor (phenbutazone) in the presence and absence of APN inhibitor (phenbutazone); CFT073 and UTI89 were first incubated with and without OmpT inhibitor (3.2 mM) for 1 h, or first incubated with and without APN inhibitor (1.0 mM) for 30 min, and then incubated with NO-AH (10 μM) in PBS at 37 °C for 2 h. (λex: 640 nm, λem: 700 / 30 nm) Scale bar: 2.5 μm. (f) Confocal images of CFT073 with NO-AH (10 μM) at scales of 10 μm and 5 μm. Data are expressed as mean ± SD (n = 3).

[0023] Figure 10The in vitro fluorescent bacteria (approximately 10 μM) of different Escherichia coli strains after co-incubation with NO-AH (10 μM) are shown. 8 Confocal microscopy analysis (CFU / mL). (ab) Confocal images of CFT073, UT89, J96, and BL21 after co-incubation with NO-AH (10 μM) in PBS at 37 °C for 2 hours; and their corresponding relative fluorescence intensities. (λex: 640 nm, λem: 700 / 30 nm.) Scale bar: 2.5 μm. Data are expressed as mean ± SD (n = 3).

[0024] Figure 11 NO-AH (10 μM) and 10 μM were shown. 4 CFU / mL to 10 8 Normalized fluorescence intensity (n = 3) after co-incubation with E. coli CFT073 in the CFU / mL range. The dashed line represents the normalized mean fluorescence intensity of NO-AH in the absence of bacteria, plus three times the standard deviation of the signal (SD).

[0025] Figure 12 shows CFT073 (approximately 10 8 CFU / mL) and Proteus mirabilis (approximately 10 CFU / mL) 8 In vitro fluorescence confocal microscopy analysis of differentiation between live cultures (CFU / mL). (a) Confocal images of different proportions of co-cultures of *Proteus mirabilis* and CFT073 after co-incubation in PBS with NO-AH (10 μM) at 37°C for 2 hours; scale bar: 2.5 μm. (b) FCM analysis of different proportions of co-cultures of *Proteus mirabilis* and CFT073 after co-incubation in PBS with NO-AH (10 μM) at 37°C for 2 hours. (c) Schematic diagram of the spatial distribution of the two enzymes in the mixed bacteria. (d) Confocal image of Hoechst-stained co-cultures of *Proteus mirabilis* and CFT073 after co-incubation in PBS with NO-AH (10 μM) at 37°C for 30 minutes. (e) Confocal image of Hoechst-stained co-cultures of *Proteus mirabilis* and CFT073, in which APN was added to the mixed bacterial environment and then co-incubated with NO-AH (10 μM) in PBS at 37°C for 30 min (Hoechst: λex: 405 nm, λem: 460 / 30 nm; NO-AH: λex: 640 nm, λem: 700 / 30 nm). Scale bars: 10 μm and 5 μm. (n = 3).

[0026] Figure 13Scatter plots showing the colocalization of red and blue pixel intensities from NO-AH and Hoechst, respectively. (a) Hoechst-stained coculture of Proteus mirabilis and CFT073 after incubation in PBS with NO-AH (10 μM) at 37°C for 30 min. (b) Hoechst-stained coculture of Proteus mirabilis and CFT073 after APN was added to a mixed bacterial environment and then incubated in PBS with NO-AH (10 μM) at 37°C for 30 min.

[0027] Figure 14 Confocal images are shown of CFT073 incubated with NO-AH (10 μM) and trypsin (1 μg / mL) for 30 min at 37 °C in PBS (pH 7.4), with and without BBI (10 μg / mL), as well as confocal images of Proteus mirabilis incubated with NO-AH and trypsin for 30 min. (λex: 640 nm, λem: 700 / 30 nm.) Scale bar: 2.5 μm.

[0028] Figure 15 The results of live / dead staining of CFT073 with NO-AH (10 μM) are shown. (a) Confocal fluorescence images of CFT073 after double staining with SYTO 9 and propidium iodide (PI) in PBS at 37°C for 2 hours, emitted at 500 nm (green channel) and 635 nm (red channel); (b) and their corresponding fluorescence intensities. λex: 488 nm. λem: 490–520 nm / 600–650 nm. Scale bar: 10 μm. SYTO 9 has a high affinity for DNA and produces green fluorescence in live bacteria, while PI indicates membrane integrity and produces red fluorescence in dead bacteria. Data are presented as mean ± SD (n = 3).

[0029] Figure 16 The cell viability of 3T3 / NIH cells after co-incubation with different concentrations of NO-AH for 24 hours is shown. Data are presented as mean ± SD (n = 3).

[0030] Figure 17 The establishment of the UTI mouse model is shown. (a) Bacterial counts of *E. coli* CFT073 in urine, bladder, urethra, and kidneys of mice 6, 24, and 48 hours after infection with *E. coli* CFT073; (b) IL-6 levels in kidneys, bladder, and urethra 6, 24, and 48 hours after infection with CFT073. Data are presented as mean ± SD (n = 3).

[0031] Figure 18 Hematoxylin and eosin (H&E) staining of the bladder, urethra, and kidneys of mice infected with Escherichia coli CFT073 at 6, 24, and 48 hours are shown. Scale bar: 100 μm or 10 μm.

[0032] Figure 19 shows in vivo near-infrared fluorescence (NIRF) imaging in live mice. (a) Schematic diagram of NIRF imaging in UTI-infected mice after intravesical injection of NO-AH (50 μM) and imaging at different time points. (bc) H&E staining and IL-6 levels in the bladder and urethra of healthy mice and UTI-infected mice 6 hours after infection. (de) NIRF images of UTI-infected and healthy mice after intravesical injection of NO-AH (50 μM) and CyOH (50 μM), respectively, and their corresponding fluorescence signals. λex: 660 nm, λem: 710 nm. (f) Schematic diagram of NIRF imaging in UTI-infected mice after intravenous injection of NO-AH (50 μM) and imaging at different time points. (gh) NIRF images of UTI-infected and healthy mice after intravenous injection of NO-AH (50 μM); and their corresponding fluorescence signals. λex: 660 nm, λem: 710 nm. In (g), the fluorescent region of infected mice at 4 hours is highlighted with a dashed circle. (i) Bacterial count of CFT073 present in urine, bladder, and urethra 2 days after bacterial infection. (j) IL-6 levels in healthy mice and UTI mice that relapsed 2 days after bacterial infection. (k) NIRF imaging of UTI-infected mice injected with NO-AH (50 μM) at 6 hours and 2 days. λex: 660 nm, λem: 710 nm. (l) In vivo detection protocol for UTI. Data are presented as mean ± SD (n = 3). Detailed Implementation

[0033] As discussed in more detail below, the inventors have developed a peptide probe according to formula (I) that utilizes the proteolytic activity of OmpT (an outer membrane protease of E. coli) and aminopeptidase N (APN) located in the periplasm of E. coli (see below). Figure 1 The peptide probes of this invention are designed to specifically target urinary tract pathogenic *Escherichia coli* through their synergistic response to OmpT and APN, enabling the detection of urinary tract infections both in vitro and in vivo. Specifically, as disclosed in the embodiments herein, the inventors have demonstrated that the peptide probes of this invention (referred to as NO-AH) exhibit extremely high selectivity for bacterial strains expressing both OmpT and APN. Furthermore, the specificity of OmpT to NO-AH results in low nonspecific background fluorescence signal in the cellular microenvironment, which facilitates sensitive signal readout. Moreover, the inventors have demonstrated that NO-AH enables real-time in vivo observation and monitoring of urinary tract bacterial infections.

[0034] Therefore, the present invention provides a peptide probe according to formula (I):

[0035] in, P does not exist or is -C(O)C 1-6 alkyl; Z represents a peptide containing the amino acid sequence according to formula (II):

[0036] in, n is an integer from 0 to 6; m is an integer from 0 to 5; Each X 1 Independently selected from any amino acid; X 3 For Arg or Lys; Each X 4 Independently selected from any amino acid; When m is 0, X 5 It does not exist or is selected from any amino acid; and When m is between 1 and 5, X 5 Selected from any amino acid; L represents a bond or a self-splitting linker; and Q is a detectable marker; or The peptide probe salt or solvate.

[0037] To avoid ambiguity, in equation (II), n can be any integer selected from 0, 1, 2, 3, 4, 5, and 6; and m can be any integer selected from 0, 1, 2, 3, 4, and 5. Similarly, to avoid ambiguity, m is 0 and X... 5 In the non-existent implementation scheme, L is X 3 The key connected to Q, or L, is X. 3 A self-splitting linker connected to Q; and where m is 1 to 5 and X 5 In the embodiments selected from any amino acid, L is X 5 The key connected to Q, or L, is X. 5 A self-splitting linker connected to Q.

[0038] The amino acid sequences disclosed herein are shown with the N-terminus on the left, and when the sequence spans multiple lines, the N-terminus is at the upper left. Within a polypeptide chain, each amino acid is linked by a peptide bond between the carboxyl group of one amino acid and the amino group of the next amino acid in the chain. A single amino acid, once linked in the polypeptide chain, is referred to as a "residue" or "amino acid residue." The amino acid sequences listed in this application are shown using the standard letter abbreviation for amino acids. In the context of this disclosure, the term "amino acid" covers any naturally occurring or non-natural amino acid. It includes amino acids of any chiral configuration. Amino acids can be, in particular, naturally occurring α-amino acids. Amino acids can be, in particular, naturally occurring L-amino acids. Amino acids can be, in particular, naturally occurring α-L-amino acids.

[0039] In embodiments herein, the word “comprising” can be interpreted as requiring the mentioned features, rather than limiting the presence of other features. Alternatively, the word “comprising” can also refer to situations where the listed components / features are intended to be present only (e.g., the word “comprising” can be replaced by the phrases “consisting of” or “substantially composed of”). It is explicitly considered that both broader and narrower interpretations can be applied to all aspects and embodiments of the invention. In other words, the word “comprising” and its synonyms can be replaced by the phrases “consisting of” or “substantially composed of” or their synonyms, and vice versa. Therefore, to avoid ambiguity, in some embodiments, as defined herein, Z in formula (I) represents a peptide comprising or composed of the amino acid sequence according to formula (II).

[0040] It should be understood here that as long as the peptide probe of the present invention contains the OmpT proteolytic site R↓(R / K) (i.e., in formula (II) -RX), it is suitable for use with respect to the OmpT proteolytic site R↓(R / K). 3 - indicates), then the flanking amino acids (in formula (II) are represented by X) 1 X 4 and X 5 The amino acid (indicated) may be present (and can be any amino acid) or absent. Flanking amino acids can confer additional beneficial functions to peptide probes, such as improved stability and / or selectivity, but are not essential for achieving the core function of peptide probes, namely the detection of urinary pathogenic Escherichia coli.

[0041] In the peptide probe of formula (I), when X is present... 1 X 4 and X 5 When, X in equation (II) 1 X 4 and X 5 Each amino acid is independently selected. For example, when X is present... 1 X 4 and X 5In this context, these amino acids can be either standard amino acids or non-standard amino acids. The term "standard amino acid" refers to the 20 amino acids encoded by the universal genetic code and are the building blocks of most proteins and peptides in organisms. The 20 standard amino acids are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). To avoid ambiguity, standard amino acids are L-amino acids. The term "non-standard amino acid" refers to amino acids that are not included in the list of the 20 amino acids considered standard amino acids. Examples of non-standard amino acids include, but are not limited to, ornithine (Orn), citrulline (Cit), 2,4-diaminobutyric acid (DABA), 3-azidoalanine, 6-azidolysine, and propargylglycine. Other examples of non-standard amino acids include the corresponding D-amino acids of standard amino acids, such as D-alanine (D-Ala) or D-phenylalanine (D-Phe). In some embodiments, in formula (II), when X is present... 1 X 4 and X 5 At that time, they were each independently selected from any standard amino acid or its D-amino acid.

[0042] In some preferred embodiments, in formula (II), each X 1 Independently selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; X 3 For Arg or Lys; each X 4 Independently selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; when m is 0, X 5 The amino acid X is absent or selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; and when m is 1 to 5, X 5 Selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro or their D-amino acids.

[0043] The peptide portion (i.e., Z in formula (I)) of the peptide probe of the present invention includes one or more basic amino acids that can impart a positive charge to the peptide probe when it is in a physiological environment (e.g., an environment with a pH of about 7.4) or in an alkaline environment (e.g., pH 7-9) that may be present in the bladder or urethra of a subject suffering from a urinary tract infection. Not wishing to be bound by theory, the inventors believe that when the peptide probe of the present invention exhibits a net positive charge, the peptide probe will interact with and bind to lipid and lipopolysaccharide (LPS) molecules present in the outer membrane of Gram-negative bacteria (e.g., Escherichia coli). Therefore, in some embodiments, in formula (II), each X 1 X 4 and / or X 5 At least one of them is independently selected from any basic amino acid, such as His, Arg, Lys, Orn, Cit and their D-amino acids (e.g., Arg or Lys). Typically, in formula (II), at least one X 1 For His, Lys, Arg, or their D-amino acids, at least one X 4 His, Lys, Arg, or their D-amino acids, and / or X 5 It is His, Lys, Arg, or their D-amino acids.

[0044] In some embodiments, the peptide moiety of the peptide probe may include one or more nonpolar amino acids, such as Gly, Ala, or Val, or amino acid sequences commonly used as spacer portions, such as repeated combinations of Gly and Ser. In some other embodiments, the peptide moiety of the peptide probe may include one or more prolines, or D-amino acids such as D-Phe. Including one or more prolines or D-amino acids in the peptide moiety of the peptide probe can improve the stability of the peptide probe to nonspecific protease digestion, thereby improving the selectivity of the peptide probe. Therefore, in some embodiments, in formula (II), at least one X 1 At least one X 4 and / or at least one X 5 It is Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Pro, or their D-amino acids. For example, in formula (II), at least one X 1 For D-Phe or at least one X 4 For D-Phe; or at least one X 1 For D-Phe and at least one X 4 For D-Phe. Typically, in equation (II), at least one X 1 It is D-Phe.

[0045] In some preferred embodiments, in formula (I), Z represents a peptide consisting of an amino acid sequence according to formula (IIa):

[0046] in, X 1 Selected from His, Lys, Arg, or their D-amino acids, X 2 ′ and X 3 Each is independently either Phe or D-Phe, and X 5 ′ represents Arg or Lys; and Where L is X 5 The key connected to Q, or L is connected to X. 5 ′ is connected to the self-splitting linker of Q.

[0047] In some embodiments, Z in the peptide probe of the present invention represents a peptide comprising an amino acid sequence according to formula (IIa), or a peptide consisting of an amino acid sequence according to formula (IIa), and X 1 ′ is Arg, and X 2 ′ and X 3 Each is D-Phe.

[0048] The N-terminus of the peptide probe of the present invention includes -C(O)C. 1-4 Alkyl groups can improve the stability of peptide probes against non-specific protease digestion, thereby increasing the selectivity of the peptide probe. Therefore, in some embodiments, in formula (I), P is -C(O)C 1-4 Alkyl groups, for example, P is an acetyl group.

[0049] In some preferred embodiments, the peptide probe of the present invention is shown in SEQ ID NO: 1:

[0050] in, L represents a bond or a self-splitting linker; and Q is a detectable marker; or It is a salt or solvate of the peptide probe.

[0051] For example, the peptide probe of the present invention can be as shown in SEQ ID NO:2:

[0052] SEQ ID NO: 2

[0053] in, L represents a bond or a self-splitting linker; and Q is a detectable marker; or It is a salt or solvate of the peptide probe.

[0054] To avoid ambiguity, in SEQ ID NO: 1 and 2, L and Q can be L and Q as defined herein for equation (I).

[0055] As used herein, the term "self-cleaving linker" refers to a linker that undergoes degradation under specific conditions, particularly after the cleavage of a peptide bond adjacent to the linker. Degradation of the linker leads to the release of a detectable label of the peptide probe. Degradation of the linker can occur, for example, via electronic cascade or via elimination pathways. A well-known example of a self-cleaving linker is an aminobenzyl alcohol group (PAB). In some embodiments of the invention, in formula (I) (and also in SEQ ID NO: 1 and 2), L can be a linker according to formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg), or (Lh):

[0056] in, Each R a Choose independently from the groups composed of O and NH. Each R b Independently selected from the groups consisting of O, NH, -OC(O)O-, and -OC(O)NH-; and This indicates the binding site between the connecting portion and Z, while This indicates the binding site between the connecting portion and Q.

[0057] In some exemplary embodiments, L is the connecting part according to the following formula: , in, This indicates the binding site between the connecting portion and Z, while This indicates the binding site between the linker and Q; this linker may also be referred to herein as the p-aminobenzyl (PAB) linker.

[0058] As used herein, the term "detectable tag" refers to a molecular tag attached to a peptide probe that is detectable when attached to the peptide probe, or detectable after being released from the peptide probe following cleavage by the proteases OmpT and APN. It should be understood that the present invention is not limited to the use of any particular type of "detectable tag," as long as the detectable tag can be attached directly or via a self-cleaving linker to the C-terminus of the peptide and can be detected after release from the peptide probe. Detection of the detectable tag can be performed by any suitable method, including fluorescence spectroscopy, any other optical method, or methods such as photoacoustic (PA) imaging. The peptide probe may not contain a self-cleaving linker when the detectable linker contains a functional group (e.g., an NH2 or OH group) that allows direct coupling to the C-terminus of the peptide. Conversely, the detectable linker can be directly attached to the Z-terminus of the peptide probe. Thus, in some embodiments, in formula (I), when Q is a detectable linker containing a functional group (e.g., an NH2 or OH group) that allows direct coupling to the C-terminus of the peptide, L is a bond.

[0059] In some embodiments, Q in formula (I) (and also in SEQ ID NO: 1 and 2) can be a fluorescent dye. For example, Q can be selected from the group consisting of coumarin, naphthimide, xanthracene (e.g., fluorescein and its analogues), rhodamine, anthocyanin, porphyrin, nitrobenzofuran, and boron-dipyrrolemethylene. In some embodiments, Q can be a near-infrared (NIR) fluorescent dye. Thus, in some preferred embodiments, Q can be selected from the group consisting of CyOH, Cy5.5, and Cy7.

[0060] In some other embodiments, in formula (I) (and also in SEQ ID NO: 1 and 2), Q can be a chemiluminescent label or a bioluminescent label. For example, Q can be selected from the group consisting of luminol, fluorescein, chemiluminescent labels based on 1,2-dioxane, and acridine esters. Clinical use of chemiluminescent or bioluminescent labels may be preferred because this allows for the detection of detectable labels by eye without the need for specialized equipment.

[0061] To avoid ambiguity, other detectable markers may be used for the peptide probes of this invention, provided that the detectable marker can be directly or via a self-cleaving linker attached to the C-terminus of the peptide and can be detected once released from the peptide probe. Examples of other detectable markers include gold nanoparticles, quantum dots, iron oxide nanoparticles, and metal complexes (e.g., copper(II)-based complexes, platinum(II)-based complexes, and ruthenium(II)-based complexes).

[0062] In some exemplary embodiments, the peptide probe of the present invention is the NO-AH disclosed in the embodiments herein, which has the following structure (SEQ ID NO: 3):

[0063] SEQ ID NO: 3.

[0064] More specifically, in some exemplary embodiments, the peptide probe is the NO-AH disclosed in the Examples section of this document, having the following structure (SEQ ID NO: 4):

[0065] SEQ ID NO: 4.

[0066] The peptide probes disclosed herein can be synthesized using methods known in the art. For example, they can be prepared by chemical synthesis methods (e.g., FMOC solid-phase peptide synthesis). Alternatively, the peptide moiety of the peptide probe (i.e., the portion represented by Z in formula (I)) can be prepared by recombinant protein production techniques, such as by recombinant expression of the peptide moiety in bacterial, yeast, insect, fungal, plant, or mammalian cells, or by cell-free in vitro peptide expression methods. Subsequently, the peptide moiety of the peptide probe can be modified by chemical synthesis methods to set a detectable label, and, when present, set an N-terminal -(O)C 1-4 Alkyl groups and self-cleaving linkers.

[0067] As used herein, the term “about” may allow for a degree of variability in a value or range, for example, within 10%, 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005%, or 0.001% of the limits of the value or range, and includes the exact value or range.

[0068] The phrase "basically composed of..." and its synonyms can be interpreted in this text as referring to materials that may contain small amounts of impurities. For example, the purity of the material can be greater than or equal to 90%, such as greater than 95%, greater than 97%, greater than 99%, greater than 99.9%, greater than 99.99%, greater than 99.999%, or 100%.

[0069] The peptide probes of the present invention can form salts or solvates. Salts suitable for the peptide probes of the present invention include those in which the counterions are pharmaceutically acceptable. However, the use of salts having non-pharmaceutical-acceptable counterions is within the scope of the present invention, for example, when the peptide probe is used only in vitro, or as an intermediate in the preparation of the peptide probe and its pharmaceutically acceptable salts and solvates and their physiologically functional derivatives. Suitable salts used according to the present invention include salts formed with organic or inorganic acids. In particular, suitable salts formed with acids used according to the present invention include salts formed with the following acids: inorganic acids, strong organic carboxylic acids, such as unsubstituted or, for example, halogenated alkane carboxylic acids of 1 to 4 carbon atoms, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids, or organic sulfonic acids, such as unsubstituted or, for example, halogenated (C 1-4 )-alkyl- or aryl-sulfonic acids. Pharmaceutically acceptable acid addition salts include salts formed from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, citric acid, tartaric acid, acetic acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, trifluoroacetic acid, succinic acid, perchloric acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, oxalic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, hydroxyethylsulfonic acid, ascorbic acid, malic acid, phthalic acid, aspartic acid, and glutamic acid, lysine, and arginine. Suitable cations that may be present in the salt include alkali metal cations, especially sodium, potassium, and calcium, as well as ammonium or amino cations. In some embodiments, the peptide probe of the present invention is in the form of a hydrochloride salt.

[0070] Those skilled in the art of organic chemistry will understand that many organic compounds can form complexes with solvents in which they react or precipitate or crystallize. These complexes are called "solvates." For example, a complex with water is called a "hydrate." Complexes may contain solvent in stoichiometric or non-stoichiometric amounts. Solvates are described in *Water-Insoluble Drug Formulation*, 2nd edition, R. Lui CRC Press, page 553 and Byrn et al ., Pharm Res 12(7), 1995, 945-954. Before being formulated in solution, the peptide probes of the present invention may be in the form of a solvate, for example, a pharmaceutically acceptable solvate. Hydrates are examples of pharmaceutically acceptable solvates.

[0071] Composition: This invention provides compositions comprising the peptide probe of this invention and at least one binder, carrier, or excipient. Preferably, the compositions of this invention are pharmaceutical compositions. For example, the composition may be a pharmaceutical composition comprising the peptide probe of this invention and at least one pharmaceutically acceptable carrier.

[0072] The compositions of the present invention are suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal, and topical (including skin, oral, sublingual, and intraocular) administration. However, the most suitable route may depend on the characteristics of the subject, such as species, age, weight, sex, medical condition, type and severity of urinary tract infection the subject has, is suspected of having, or is at risk of developing, and other relevant medical and physical factors. Preferably, the compositions of the present invention are suitable for administration by injection and / or infusion, such as intravascular or transurethral injection.

[0073] Pharmaceutically acceptable binders, carriers, or excipients suitable for inclusion in the pharmaceutical compositions of the present invention may be selected based on the intended route of administration and standard pharmaceutical practice. These pharmaceutically acceptable binders, carriers, or excipients may be chemically inert to the active compound and may not cause harmful side effects or toxicity under the conditions of use. Suitable pharmaceutical compositions may be referenced, for example, in Remington, *The Science and Practice of Pharmacy*, 19. th ed., Mack Printing Company, Easton, Pennsylvania (1995). Additionally, those skilled in the art can routinely prepare suitable formulations using conventional techniques and / or according to standard and / or acceptable pharmaceutical practices. It should be understood that, in addition to the components specifically mentioned above, compositions used in this invention may include other reagents conventional in the art and relevant to the type of composition discussed. In some embodiments, the composition comprises the peptide probe of this invention and a pharmaceutically acceptable carrier, such as water for injection. The compositions of this invention may also comprise pharmaceutically acceptable buffers (e.g., sodium citrate, phosphate-buffered saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer, and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer), and / or one or more selected from the group consisting of polyethylene glycol (e.g., PEG 400), nonionic detergents (e.g., polysorbates, such as polysorbate-20), and sugars (e.g., cyclodextrin).

[0074] Practicality: This invention provides a method for detecting urinary tract infection in a urine sample obtained from a subject, the method comprising: a) Provide a urine sample obtained from the subject; b) Contact the urine sample with the peptide probe according to the invention; c) Determine the presence of urinary tract pathogenic Escherichia coli in the urine sample by detecting the detectable markers of the peptide probe in the urine sample.

[0075] To avoid ambiguity, each step in steps a) through c) of this method is performed in vitro. Therefore, the method for detecting urinary tract infections in urine samples obtained from subjects can be considered an in vitro method.

[0076] Steps a) to c) of the method of the present invention can be repeated using one or more different urine samples obtained from the subject. For example, one or more urine samples obtained over several hours, days, weeks, or months. For example, steps b) to c) can be performed using a urine sample obtained from the subject before administering a dose of appropriate treatment for a urinary tract infection, and then repeated using a urine sample obtained from the subject after administering a dose of appropriate treatment for a urinary tract infection, for example, 1 to 7 days, 1 to 3 weeks, or 1 to 3 months after administering a dose of appropriate treatment for a urinary tract infection.

[0077] As used herein, the terms “subject” or “patient” are well known in the art and are used interchangeably herein, referring to mammals, including dogs, cats, rats, mice, monkeys, cattle, horses, goats, sheep, pigs, camels, and most preferably, humans. The term does not indicate a specific age or sex. Therefore, it is intended to cover adult and newborn subjects, whether male or female. In embodiments, the subject is known or suspected of having a urinary tract infection, and / or is known or suspected of being at risk of developing a urinary tract infection, particularly one caused by urinary tract pathogenic Escherichia coli. In some embodiments, the subject is a subject with a disease or condition known to increase the risk of developing a urinary tract infection and / or increasing the risk of developing a more severe form of urinary tract infection. For example, the subject may be a subject known or suspected of having a disease or condition commonly associated with urinary tract infections, such as diabetes, hypertension, chronic kidney disease, dyslipidemia, ischemic heart disease, prostate problems, and impaired immune function.

[0078] Step c) can be performed using any technique suitable for detecting the detectable label present in the peptide probe of the present invention, as long as the label is released from the peptide probe by the proteolytic activity of OmpT and APN. For example, when the detectable label present in the peptide probe used in step c) is a fluorescent dye (e.g., an NIR fluorescent dye, such as CyOH), step c) may include the use of fluorescence spectroscopy. In embodiments where the detectable label is a chemiluminescent label or a bioluminescent label, step c) can be performed visually (e.g., by a clinician or any other healthcare professional) without the need for specialized equipment.

[0079] Therefore, upon contact with a urine sample, the detection and / or quantification of the detectable labeling of the peptide probe of the present invention can be used to guide clinicians in determining appropriate disease monitoring procedures, appropriate treatments, or to inform clinicians that the subject is in remission of a urinary tract infection.

[0080] Therefore, this article also provides a method for determining one or more suitable therapeutic agents for treating a subject's urinary tract infection, the method comprising: i) Provide a urine sample obtained from the subject; ii) Contact the urine sample with the peptide probe according to the invention; iii) Determining the presence of urinary tract pathogenic Escherichia coli in the urine sample by detecting the detectable marker of the peptide probe in the urine sample; and iv) Based on the detection of detectable markers of peptide probes in urine samples, identify one or more suitable therapeutic agents for the treatment or prevention of urinary tract infections in the subject.

[0081] As is evident from the above disclosure, the peptide probe of the present invention can also be used in methods for treating or preventing urinary tract infections in subjects. Such methods may include: A. Provide a urine sample obtained from the subject; B. Contact the urine sample with the peptide probe according to the invention; C. To determine the presence of urinary tract pathogenic Escherichia coli in the urine sample by detecting the detectable markers of the peptide probe in the urine sample; and D. Based on the detection of detectable markers of the peptide probe in the urine sample, administer a dose of one or more therapeutic agents for treating urinary tract infections to the subject.

[0082] Methods for treating or preventing urinary tract infections may include administering a peptide probe according to the invention to a subject, for example by urethral or intravenous injection, followed by detecting a detectable marker of the peptide probe in the subject's bladder, urethra, and / or urine, and then administering a dose of one or more therapeutic agents for treating urinary tract infections to the subject.

[0083] The peptide probe of the present invention can also be used in methods for determining one or more suitable therapeutic agents for treating a subject with a urinary tract infection. This method may include, for example, administering the peptide probe according to the invention to a subject by transurethral or intravenous injection, and determining one or more suitable therapeutic agents for treating the urinary tract infection by detecting a detectable marker of the peptide probe in the subject's bladder, urethra, and / or urine.

[0084] In methods for treating or preventing urinary tract infections in a subject, or in methods for determining one or more suitable therapeutic agents for treating a urinary tract infection in a subject, the step of detecting a detectable marker of the peptide probe can be performed using any suitable detection means. For example, the detectable marker can be detected in the bladder and / or urethra using in vivo imaging techniques. For instance, when the detectable marker is an NIR fluorescent dye (e.g., CyOH), the detectable marker can be detected in the subject's bladder and / or urethra using in vivo near-infrared fluorescence (NIRF) imaging. Alternatively or optionally, the detectable marker can be detected in the urine excreted by the subject after the peptide probe has been administered to the subject's bladder.

[0085] Suitable therapeutic agents for the treatment or prevention of urinary tract infections include, but are not limited to, antimicrobial agents active against urinary tract pathogenic Escherichia coli. For example, suitable therapeutic agents may include antibiotics selected from the group consisting of trimethoprim, sulfamethoxazole, fosfomycin, nitrofurantoin, β-lactam-based cephalexin, ceftriaxone, cefaclor, tetracyclines, and antimicrobial peptides (such as polymyxins B and E, and vancomycin). The dosage of the suitable therapeutic agent required to achieve a therapeutic effect will vary depending on the specific route of administration and the characteristics of the subject receiving treatment, such as species, age, weight, sex, medical condition, severity of urinary tract infection, and other relevant medical and physical factors. Physicians with general skills can readily determine and administer the effective dosage of the suitable therapeutic agent required for the treatment or prevention of urinary tract infections.

[0086] Reagent test kit: The present invention also provides an in vitro diagnostic kit for detecting, screening, monitoring, classifying, selecting subjects for treatment of urinary tract infections, determining the effectiveness of treatment in subjects with urinary tract infections, and / or predicting urinary tract infections in subjects. The kit comprises one or more peptide probes according to the present invention, or comprises a composition of the present invention. For example, the kit may comprise a peptide probe referred to herein as NO-AH.

[0087] In some embodiments, the kit of the present invention comprises one or more carriers (e.g., buffer solutions) and optionally one or more therapeutic agents suitable for treating or preventing urinary tract infections. Examples of such therapeutic agents include those described herein.

[0088] In embodiments where the peptide probes present in the kit of the present invention contain a chemiluminescent or bioluminescent label, the kit may further include components required to detect the label. For example, if the peptide probes present in the kit contain luciferin as a detectable label, the kit may include luciferase.

[0089] In some embodiments, the kit includes one or more containers and may also include sampling devices such as bottles, bags, vials, syringes, and test tubes. Other components may include needles, diluents, washing reagents, and buffers. Typically, the kit includes instructions, such as those instructing the user to mix a specified amount of the peptide probe or composition of the present invention with a specified amount of diluent.

[0090] To avoid ambiguity, the peptide probes or compositions of the present invention, and optional diluents, are present in the kits according to the present invention in the following forms and amounts suitable for using the peptide probes in methods for detecting, screening, monitoring, classifying, selecting treatment for urinary tract infections in subjects, determining whether treatment is effective in subjects with urinary tract infections, and / or predicting urinary tract infections in subjects. Those skilled in the art can readily determine the amounts of the peptide probes or compositions of the present invention and optional diluents suitable for use according to the present invention.

[0091] Other aspects of the invention are defined in the following numbered clauses: §1. A probe comprising: a) At least 5 amino acid residues; b) Self-splitting connectors; and c) Dyes that exhibit fluorescence only in their free form. The two amino acid residues adjacent to the self-cleaving linker are derived from dibasic amino acids (e.g., Arg and Lys).

[0092] §2. According to the probe of §1, wherein the at least 5 amino acid residues include the sequence (arginine, phenylalanine, phenylalanine, arginine, arginine-SEQ ID NO: 6).

[0093] §3. According to the probe of §1 or §2, wherein the self-cleaving linker comprises 4-aminobenzyl alcohol.

[0094] §4. The probe according to any one of §1 to §3, wherein the dye comprises a hemicyanine dye.

[0095] The contents of articles, patents, and patent applications mentioned or cited herein, as well as all other documents and electronically available information, are incorporated herein by reference in their entirety, to the same extent that each individual publication is expressly and separately incorporated herein by reference. The applicant reserves the right to incorporate any and all material and information entities from any such articles, patents, patent applications, or other physical and electronic documents into this application.

[0096] Other aspects and embodiments of the invention will now be discussed with reference to the following non-limiting examples.

[0097] Example

[0098] As disclosed herein, the inventors have developed a near-infrared OmpT-APN hemicyanin fluorescent probe (referred to as NO-AH) for real-time in vitro and in vivo bacterial imaging of urinary tract infections. Figure 1 As shown, NO-AH contains a hemicyanine signaling moiety that is doubly locked by a dual protease-activated moiety, which is sequentially cleaved by OmpT and then by aminopeptidase N (APN). A self-cleaving linker (4-aminobenzyl alcohol; PABA) between the hemicyanine reporter molecule (CyOH) and the peptide sequence facilitates the release of free CyOH during the sequential cleavage by both proteases via a 1,6-elimination reaction. In UTIs, pathogens (such as urinary tract pathogenic Escherichia coli) colonize the host's periurethral region and urethra, then ascend the urethra to the bladder. Urinary tract pathogenic Escherichia coli (such as CFT073 and UTI89) are known to express OmpT and APN. Therefore, the release of CyOH from the peptide probe and its subsequent detection can confirm the presence of urinary tract pathogenic Escherichia coli in the host.

[0099] Material: Chemicals, solvents and culture media Unless otherwise specified, all chemicals, solvents, and other reagents were used without further purification. Fmoc-protected amino acids and benzotriazole tetramethyluranium hexafluorophosphate (HBTU) were purchased from Jier Biochemical (Shanghai) Co., Ltd. N,N-diisopropylethylamine (DIPEA), phosphorus tribromide (PBr3), trifluoroacetic acid (TFA), anhydrous dichloromethane, anhydrous acetonitrile, and anhydrous tetrahydrofuran were purchased from Sigma Aldrich. 4-Aminobenzyl alcohol (PABA), N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), and IR775-chloride were purchased from TCI Corporation of Japan and distributed by Sihai Chemical Pte Ltd. Pte. Phenylephrine (HY-B0134-10 mg) was purchased from MedChemExpress and distributed by Bio.Etc. Pte Ltd. Luria-Bertani (LB) broth, tryptone soybean broth (TSB), brain and heart infusion (BHI) broth, and bacterial culture agar were purchased from Beckton-Dickinson Ltd. (Singapore) and distributed by Yuli Pharmaceutical Ltd. Dulbecco modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin, trypsin-EDTA, and the LIVE / DEAD™ BacLight™ bacterial viability kit (L7012) were purchased from Thermo Fisher Scientific Ltd.

[0100] Enzymes, bacterial strains, cell lines, and mice

[0101] Escherichia coli BL21 (DE3) was purchased from New England Biolabs Pte Ltd (Singapore). Wild-type urinary tract pathogenic Escherichia coli UTI89 was a gift from Professor Kimberley Kline's laboratory (Singapore Centre for Environmental and Life Sciences Engineering, Nanyang Technological University). Escherichia coli CFT073 ATCC® 700928™ and Escherichia coli J96 (ATCC® 700336™) were purchased from ATCC (USA) and supplied by Chemoscience Pte Ltd. Methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis (MRSA) were also present. E. faecalis OG1RF and Proteus mirabilis ( P. mirabilisThe following were donated by Professor Qiao Yuan of Nanyang Technological University: Mouse embryonic fibroblast cell line (NIH / 3T3) purchased from the American Type Culture Collection (ATCC); Recombinant OmpT donated by Professor Bo Liedberg's team; Lysozyme, β-galactosidase (β-Gal; G4155-1KU), nitroreductase (NTR), NADH, caspase 3 (Casp3), and glutathione (GSH) purchased from Sigma-Aldrich; Recombinant mouse cathepsin B (CF, 10 μg) purchased from R&D Systems and distributed by SingLab Technologies Pte Ltd; Aminopeptidase N kit (ab273292) purchased from Abcam Singapore Pte Ltd; and female BALB / c mice (9-10 weeks old) purchased from SLAC (Shanghai).

[0102] instrument

[0103] Liquid chromatography-mass spectra were obtained using a Thermo Finnigan LCQ Fleet mass spectrometer. ¹H and 13 C10 NMR spectra were acquired using a Bruker Advance III 400 MHz NMR, Bruker Avance II 600 MHz NMR, or Bruker Avance II 700 MHz NMR spectra. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis was performed using a Shimadzu HPLC system equipped with an Alltima C-18 (250 × 10 mm) column at a flow rate of 3.0 mL / min, with CH3CN (containing 0.1% TFA) and water (containing 0.1% TFA) as the eluent. RP-HPLC purification was performed using a Shimadzu HPLC system equipped with an Agilent 5 prep-C18 (50 × 21.2 mm) column at a flow rate of 5.0 mL / min, with CH3CN (containing 0.1% TFA) and water (containing 0.1% TFA) as the eluent. Absorbance and fluorescence spectra were obtained using a TECAN Spark (Mennedorf, Switzerland) multimode microplate reader. Cell viability was determined using a Thermo Scientific Varioskan™ LUX multimode microplate reader. Confocal laser scanning microscopy images were acquired using a Carl Zeiss LSM 800 confocal laser microscope (Germany). Flow cytometry analysis was performed using a BD LSRFortessa X-20 flow cytometer. Animal fluorescence imaging was performed using an IVIS Lumina II (CaliperLife Sciences) imaging system.

[0104] Chemical synthesis and characterization: Synthesis of R-CyOH The synthesis of R-CyOH follows Figure 2 The synthesis method shown is performed. Further details include the following: Synthesis of CyOH CyOH was synthesized according to the reported protocol (R. Yan, Y. Hu, F. Liu, S. Wei, D. Fang, AJ Shuhendler, H. Liu, HY Chen, D. Ye, J. Am Chem Soc, 2019, 141, 10331). In a round-bottom flask, resorcinol (110.1 mg; 1.0 mmol) was first dissolved in acetonitrile (CH3CN; 3 mL). Then, K2CO3 (138.2 mg; 1.0 mmol) was added, and the mixture was stirred at 35 °C for 20 min under N2 atmosphere. Next, a solution of IR775-chlorinated CH3CN (5 mL) was added to the round-bottom flask via syringe, and the mixture was stirred at 50 °C for 6 h. Subsequently, the solution was evaporated under reduced pressure, and the crude product was purified by silica gel chromatography using CH2Cl2 / 0-10% CH3OH as the eluent to give CyOH (250 mg; 65%) as a blue-green solid. 1 H NMR (400 MHz, methanol-d4) δ 8.48 (d, J =14.1 Hz, 1H), 7.60 (s, 1H), 7.49 (dd, J = 7.4, 2.8 Hz, 1H), 7.38 (d, J = 6.5Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H), 6.74 (dd, J = 8.8, 2.3 Hz, 1H), 6.60 (t, J= 3.6 Hz, 1H), 6.06 (d, J = 14.1 Hz, 1H), 3.59 (s, 3H), 2.75 (d, J = 5.9 Hz, 2H), 2.68 (d, J = 6.7 Hz, 2H), 1.90 (s, 2H), 1.75 (s, 6H). 13C10 NMR (100 MHz, methanol-d4) δ 174.9, 172.0, 162.6, 157.5, 143.2, 140.5, 139.7, 139.4, 129.6, 128.3, 124.2, 121.9, 121.4, 121.2, 114.9, 114.5, 109.8, 102.2, 97.9, 48.6, 29.9, 28.0, 27.3, 23.9, 20.7. ESI-MS: calculated m / z values, ... 26 H 26 NO2 [M-Cl] + 384.20; Measured value: 384.29. HRMS (ESI): Calculated m / z value, C 26 H 26 NO2: 384.1964 [M-Cl] + ; Measured value: 384.1966.

[0105] Synthesis of Fmoc-Arg(Pbf)-PABA (Compound 1) First, Fmoc-Arg(Pbf)-OH (260 mg; 0.4 mmol) was dissolved in anhydrous CH2Cl2 (5 mL), and then EEDQ (396 mg; 1.6 mmol) was added to the solution. The mixture was stirred at room temperature under a N2 atmosphere for 30 minutes. After 30 minutes, PABA (197 mg; 1.6 mmol) was dissolved in anhydrous CH2Cl2 (2 mL) and added via syringe. The mixture was stirred at room temperature under a N2 atmosphere for 16 hours. After completion, the solvent was evaporated under reduced pressure. Purification by reversed-phase HPLC yielded pure compound 1 (110 mg; 36%) as a white solid. 1H NMR(400 MHz, methanol-d4) δ 7.78 (d, J = 7.3 Hz, 2H), 7.65 (t, J = 7.7 Hz, 2H), 7.54(d, J = 8.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 8.5 Hz, 4H), 4.56(s, 2H), 4.41 (d, J = 5.1 Hz, 2H), 4.21 (t, J = 6.6 Hz, 2H), 3.20 (t, J = 6.6Hz, 2H), 2.95 (s, 2H), 2.56 (s, 3H), 2.49 (s, 3H), 2.05 (s, 3H), 1.80 (d, J =13.7 Hz, 1H), 1.69 (dd, J = 9.0, 4.8 Hz, 1H), 1.42 (s, 6H). 13 C10 NMR (100 MHz, methanol-d4) δ 157.1, 143.9, 143.7, 141.2, 137.5, 137.1, 129.4, 127.4, 127.2, 126.8, 124.8, 120.0, 119.5, 66.4, 63.4, 55.2, 42.4, 29.2, 27.2, 18.2, 16.9, 11.1. ESI-MS: calculated m / z values, ... 41 H 47 N5O7S [M+H] + 754.32; Measured value: 754.46. HRMS (ESI): Calculated m / z value, C 41 H 47 N5O7S: 754.3274 [M+H] + ; Measured value: 754.3282.

[0106] Synthesis of Fmoc-Arg(Pbf)-PAB-CyOH (Compound 2)Compound 1 (52.8 mg; 0.07 mmol) was dissolved in anhydrous THF and cooled in an ice bath under N2 atmosphere. PBr3 (13.3 µL; 0.14 mmol) was slowly added dropwise using a syringe. The mixture was stirred for 2 hours. After completion, the mixture was concentrated under reduced pressure. The resulting crude product was dissolved in ethyl acetate (30 mL), washed with saturated sodium bicarbonate (30 mL × 3), then washed with brine (30 mL × 1), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was used immediately for the next reaction without further purification. Then, CyOH (6.7 mg; 0.0175 mmol) and N,N-diisopropylethylamine (DIPEA, 12 µL, 0.07 mmol) were first dissolved in anhydrous CH3CN at 55 °C and stirred for 30 minutes under N2 atmosphere. The crude product (57 mg; 0.07 mmol) was then added, and the reaction mixture was stirred at 55 °C for 6 hours under a N2 atmosphere. After completion, the resulting mixture was evaporated under pressure. Purification by reversed-phase HPLC yielded pure compound 2 (9.7 mg; 51%) as a blue solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.88(d, J = 7.7 Hz, 3H), 7.73 (t, J = 7.0 Hz, 4H), 7.66 (t, J = 8.7 Hz, 3H), 7.59(t, J = 7.8 Hz, 1H), 7.53 (dd, J = 10.9, 8.4 Hz, 2H), 7.40 (dd, J = 8.3, 5.5Hz, 4H), 7.31 (h, J = 3.9 Hz, 3H), 7.24 (d, J = 8.4 Hz, 1H), 5.38 (d, J = 5.4Hz, 1H), 4.27 (t, J = 6.1 Hz, 2H), 4.21 (t, J = 7.2 Hz, 2H), 4.13 (q, J = 8.3Hz, 2H), 3.11 – 3.01 (m, 2H), 2.91 (s, 3H), 2.68 (dt, J = 20.0, 6.2 Hz, 2H), 2.46 (s, 5H), 2.40 (s, 5H), 1.97 (s, 3H), 1.96 (t, J = 2.6 Hz, 2H), 1.88 (dd,J = 9.1, 5.8 Hz, 1H), 1.81 (t, J = 5.9 Hz, 1H), 1.74 (s, 2H), 1.38 (s, 6H). 13CNMR (100 MHz, DMSO-d6) δ 178.3, 171.5, 171.2, 161.8, 160.6, 159.3, 158.9,158.6, 158.2, 157.9, 156.5, 154.1, 144.3, 144.2, 142.7, 142.5, 141.2, 139.3,138.0, 137.8, 134.5, 133.2, 132.9, 131.9, 131.5, 130.3, 130.0, 129.2, 128.1,127.5, 127.4, 125.8, 124.8, 123.0, 120.6, 119.7, 119.4, 117.4, 116.7, 115.9, 114.5, 114.2, 113.6, 86.8, 70.1, 66.8, 66.1, 63.0, 55.6, 50.8, 47.1, 42.9, 35.0, 33.1, 29.7, 29.0, 28.9, 28.7, 27.7, 26.2, 19.4, 18.1, 12.7. ESI-MS: m / z calculated values, C 67 H 71 N6O8S [M-Cl] + 1119.50; Measured value: 1119.57. HRMS (ESI): Calculated m / z value, [C 67 H 71 N6O8S] + Cl - 1155.4821 [M+H]+; Measured value: 1155.4823.

[0107] Synthesis of Arg-PAB-CyOH (compound 3; R-CyOH) First, pure compound 2 (9.7 mg; 0.01 mmol) was dissolved in trifluoroacetic acid (TFA) / CH₂Cl₂ (4 mL, 50% v / v) in an ice bath with stirring for 2 hours. The reaction was continuously monitored by analytical HPLC. After completion, the reaction mixture was diluted with CH₂Cl₂ (20 mL) and washed with saturated sodium bicarbonate (30 mL × 2). The organic phase was then dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give a blue crude solid. The blue crude solid was used immediately in the next reaction without further purification. The crude product was dissolved in DMF (1.5 mL) and piperidine (500 μL; 5% v / v DMF solution) was added. The reaction mixture was stirred at room temperature for 20 minutes and then purified by reversed-phase HPLC to give pure compound 10 (2 mg; 31%). 1H NMR (600 MHz, methanol-d4) δ 8.74 (d, J = 15.5 Hz, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1H), 7.56 - 7.51 (m, 2H), 7.49 (d,J = 8.7 Hz, 2H), 7.46 (d, J = 3.1 Hz, 1H), 7.44 (d, J = 4.1 Hz, 1H), 7.35 (s,1H), 7.07 (d, J = 2.6 Hz, 1H), 7.02 (dd, J = 8.8, 2.4 Hz, 1H), 6.49 (d, J =15.3 Hz, 1H), 4.09 (t, J = 6.6 Hz, 1H), 3.85 (s, 3H), 3.24 (t, J = 7.2 Hz,2H), 3.04 - 2.97 (m, 1H), 2.76 (t, J = 6.7 Hz, 2H), 2.71 (t, J = 6.4 Hz, 2H), 2.69 (s, 1H), 2.04 - 1.88 (m, 6H), 1.81 (s, 6H). 13 C NMR (100 MHz, methanol-d4) δ 179.7, 163.6, 163.2, 162.9, 155.8, 147.1, 143.7, 143.4, 139.1, 134.8, 130.2, 130.0, 129.6, 128.7, 128.4, 123.6, 121.3, 119.7, 117.3, 116.8, 115.7, 115.2, 113.7, 105.0, 102.7, 71.4, 54.7, 52.0, 45.7, 41.8, 32.8, 30.1, 29.9, 28.2, 25.4, 25.0, 23.7, 23.1, 21.6. ESI-MS: m / z calculated values, C 39 H 45 N6O3 [M-Cl] + 645.35; Measured value: 645.40. HRMS (ESI): Calculated m / z value, [C 39 H 45 N6O3] + Cl -: 681.3320 [M+H] + ; Measured value: 681.3326.

[0108] Synthesis of NO-AH (compound 7): By coupling the NIR hemicyanine moiety CyOH to the OmpT-APN peptide substrate (see...) Figure 1 and 3 The fluorescent peptide probe (NO-AH, compound 7) was prepared. In short, the OmpT-APN peptide was synthesized via solid-phase peptide synthesis. Then, NIR hemicyanine (CyOH) was linked to the peptide sequence using a self-cleaving p-aminobenzoic acid (PABA) linker. The final product, the NO-AH probe, was purified by high-performance liquid chromatography (HPLC) and further characterized by mass spectrometry and nuclear magnetic resonance. Further details are provided below: Synthesis of Ac-R(Pbf)ffR(Pbf)R(Pbf)-OH (Compound 4) The peptide Ac-R(Pbf)ffR(Pbf)R(Pbf)-OH was synthesized via solid-phase peptide synthesis (SPPS) based on standard Fmoc on a 2-chlorotriphenylmethylchloro resin. The coupling reaction between two amino acids and acetic acid was carried out for 2 to 4 hours at room temperature and under a nitrogen atmosphere, using HBTU as a coupling agent and DIPEA as a basic catalyst. The reaction lasted 4 hours when coupled with Arg, and 2 hours for D-Phe and acetic acid. The Fmoc protecting group was removed for 20 minutes with a solution of 20% piperidine in dimethylformamide (DMF). The product was then cleaved from the resin using a solution of 5% TFA in dichloromethane (DCM) for 30 minutes and precipitated in cold diethyl ether to give compound 4. Compound 4 was then used without further purification. 11H NMR (400 MHz, DMSO-d6) δ 8.19 (dd, J = 16.4, 7.9 Hz, 4H), 7.99 (dd, J = 15.0, 7.8 Hz, 2H), 7.21 (d, J = 6.0 Hz, 5H), 7.16 (d, J = 3.1 Hz, 5H), 4.54 (q, J = 8.2 Hz, 1H), 4.48 (ddd, J = 12.3, 8.4, 3.8 Hz, 1H), 4.25 (td, J = 8.5, 5.0 Hz, 1H), 4.17 (q, J = 6.3, 5.1 Hz, 1H), 4.16 - 4.07 (m, 1H), 3.03 (dt, J = 9.7, 5.1 Hz, 4H), 2.95 (d, J = 6.7 Hz, 9H), 2.89 (s, 1H), 2.89 - 2.79 (m, 1H), 2.63 (dd, J = 13.9, 10.7 Hz, 2H), 2.48 (d, J = 8.0 Hz, 6H), 2.46 - 2.39 (m, 9H), 2.04 - 1.97 (m, 3H), 1.83 (s, 3H), 1.77 - 1.53 (m, 4H), 1.46 (s, 2H), 1.39 (s, 18H), 1.31 - 1.17 (m, 4H). 13 13C NMR (100 MHz, DMSO-d6) δ 173.7, 172.0, 171.8, 171.4, 171.0, 170.0, 159.4, 159.0, 158.6, 158.3, 158.0, 156.5, 156.4, 138.2, 137.9, 137.8, 137.8, 134.6, 134.6, 134.5, 131.9, 129.7, 128.5, 128.4, 126.7, 126.6, 124.8, 117.1, 116.8, 114.2, 111.3, 86.8, 65.4, 54.8, 54.2, 52.7, 52.2, 42.9, 38.2, 37.6, 30.0, 29.7, 28.7, 22.8, 19.4, 19.4, 18.1, 18.0, 15.6, 12.7, 12.7. ESI-MS: calculated m / z, C 77 H 106 N 14 O16 S3 [M+H] + 1579.71; Measured value: 1579.75. HRMS (ESI): Calculated m / z value, C 77 H 106 N 14 O 16 S3: 1579.7152 [M+H] + ; Measured value: 1579.7164.

[0109] Synthesis of Ac-R(Pbf)ffR(Pbf)R(Pbf)-PABA (Compound 5) Compound 4 (790 mg; 0.5 mmol) and N -Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline EEDQ (495 mg; 2.0 mmol) was dissolved in anhydrous DCM (5 mL) and stirred at room temperature under N2 atmosphere for 30 min. Subsequently, 4-aminobenzyl alcohol (PABA) (246 mg; 2.0 mmol) was dissolved in anhydrous DCM and added dropwise to the mixture via syringe at room temperature under N2 atmosphere. The reaction mixture was stirred for 16 h. After the reaction, the solvent was evaporated under reduced pressure, and the precipitate was collected in cold diethyl ether to give compound 5 (800 mg; 95%) as a white solid. Compound 5 was used without further purification. ESI-MS: calculated m / z, C 84 H 113 N 15 O 16 S3 [M+H] + 1684.77; Measured value: 1684.77. HRMS (ESI): Calculated m / z value, C 84 H 113 N 15 O 16 S3: 1684.7730 [M+H] + ; Measured value: 1684.7708.

[0110] Synthesis of Ac-R(Pbf)ffR(Pbf)R(Pbf)-PAB-CyOH (Compound 6)Compound 5 (168.5 mg; 0.1 mmol) was dissolved in anhydrous THF and cooled in an ice bath. PBr3 (19 μL; 0.2 mmol) was then slowly added dropwise, and the reaction mixture was stirred at 0 °C for 2 hours under N2 atmosphere. The solvent was then removed under reduced pressure. The resulting crude product was dissolved in DCM (100 mL) and washed with saturated sodium bicarbonate (50 mL × 3) and brine (50 mL × 1). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude product was then precipitated with cold diethyl ether (20 mL × 2) to give a grayish-white solid. The crude solid was then used immediately for subsequent steps without further purification. CyOH (9.6 mg; 0.025 mmol) was first dissolved in anhydrous CH3CN (1 mL), and then K2CO3 (14 mg; 0.1 mmol) was added to this solution. The reaction mixture was stirred at room temperature for 10 minutes under N2 atmosphere. A grayish-white crude solid (175 mg; 0.1 mmol) was then added to the reaction mixture, and the mixture was stirred at 50 °C for 6 hours. Afterward, the resulting mixture was evaporated under pressure. The crude solid was dissolved in DCM (50 mL) and washed with deionized water (25 mL × 2) and brine (25 mL × 1). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Purification by reversed-phase HPLC yielded pure compound 6 (25 mg, 48%) as a deep blue solid. 11H NMR (400 MHz, methanol-d4) δ 8.64 (dd, J = 14.7, 4.0 Hz, 1H), 7.78 (dd, J = 33.8, 8.5 Hz, 2H), 7.63 (d, J = 7.7 Hz, 1H), 7.49 (s, 3H), 7.47 - 7.36 (m, 3H), 7.28 (s, 1H), 7.16 (s, 3H), 7.12 (d, J = 7.5 Hz, 1H), 7.05 (t, J = 7.7 Hz, 2H), 6.96 (q, J = 8.6, 6.7 Hz, 4H), 6.44 (dd, J = 14.9, 3.0 Hz, 1H), 5.22 (d, J = 13.6 Hz, 2H), 4.61 - 4.32 (m, 5H), 4.15 (d, J = 9.7 Hz, 2H), 3.82 (s, 3H), 3.10 - 2.95 (m, 4H), 2.94 (s, 9H), 2.89 (d, J = 21.3 Hz, 2H), 2.74 - 2.62 (m, 4H), 2.55 (d, J = 2.9 Hz, 6H), 2.50 (s, 9H), 2.04 (d, J = 4.1 Hz, 9H), 2.01 (s, 3H), 1.92 (s, 2H), 1.90 (s, 3H), 1.76 (s, 6H), 1.62 - 1.49 (m, 4H), 1.41 (s, 18H), 1.32 - 1.25 (m, 4H). 13C NMR (100 MHz, methanol-d4) δ 178.2, 172.0, 162.1, 159.3, 158.9, 154.2, 145.6, 142.3, 141.9, 138.1, 137.2, 133.4, 132.6, 129.0, 128.8, 128.6, 128.3, 128.2, 128.1, 128.0, 127.1, 127.0, 126.6, 124.8, 122.4, 120.1, 117.2, 116.9, 115.8, 114.2, 114.1, 112.3, 103.6, 101.4, 94.9, 86.5, 70.2, 56.7, 54.0, 50.5, 48.3, 48.2, 48.1, 48.0, 47.9, 47.8, 47.7, 47.6, 47.5, 47.4, 47.0, 42.5, 36.5, 31.4, 29.4, 28.6, 27.3, 26.9, 23.5, 21.1, 20.2, 18.3, 17.1, 11.2. ESI-MS: m / z calculated values, C 110 H 137 N 16 O 17 S3 [(M-Cl)+2H] 2+ 1025.48; Measured value: 1025.87. HRMS (ESI): Calculated m / z value, [C 110 H 137 N 16 O 17 S3] + Cl - : 2085.9277 [M+H] + ; Measured value: 2085.9270.

[0111] Synthesis of Ac-RffRR-PAB-CyOH (Compound 7; NO-AH) First, compound 3 (25 mg; 0.01 mmol) was dissolved in DCM (1125 μL; 22.5% v / v) and cooled in an ice bath. Triethylsilane (125 μL, 2.5% v / v) was added to the reaction mixture, followed by TFA (3750 μL, 75% v / v). The reaction was stirred in an ice bath for 16 hours. The reaction was monitored by analytical HPLC. After the reaction was complete, the solvent was evaporated under reduced pressure. Purification by reversed-phase HPLC yielded pure compound 4 (10 mg; 77%) as a deep blue solid. 11H NMR (700 MHz, methanol-d4) δ 8.74 (dd, J = 25.0, 15.5 Hz, 1H), 7.75 - 7.64 (m, 2H), 7.54 (d, J = 5.0 Hz, 1H), 7.51 - 7.47 (m, 1H), 7.48 - 7.44 (m, 2H), 7.40 - 7.35 (m, 1H), 7.30 (q, J = 10.1, 8.9 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.26 - 7.23 (m, 2H), 7.22 (d, J = 3.9 Hz, 2H), 7.15 (t, J = 7.3 Hz, 2H), 7.11 (dd, J = 7.6, 4.9 Hz, 2H), 7.10 - 7.06 (m, 1H), 7.04 - 6.98 (m, 2H), 6.50 (dt, J = 15.5, 4.6 Hz, 1H), 3.86 (s, 2H), 3.07 - 2.99 (m, 5H), 2.78 (t, J = 6.6 Hz, 2H), 2.72 (t, J = 6.3 Hz, 2H), 2.08 - 2.03 (m, 2H), 1.99 - 1.91 (m, 8H), 1.82 (s, 3H), 1.81 (s, 6H), 1.71 - 1.59 (m, 4H), 1.45 (s, 2H), 1.29 (s, 3H), 1.27 - 1.09 (m, 2H), 1.05 (dd, J = 7.5, 2.2 Hz, 2H). 13C NMR (150 MHz, methanol-d4) δ 179.7, 173.8, 172.1, 163.6, 158.6, 155.7, 147.0, 143.4, 138.2, 134.8, 130.4, 130.3, 130.2, 130.2, 130.1, 130.0, 129.7, 129.6, 129.5, 129.4, 129.4, 129.2, 128.6, 128.4, 127.9, 127.8, 123.6, 121.6, 121.5, 121.4, 117.3, 115.4, 115.3, 113.7, 105.0, 102.6, 71.5, 58.0, 56.7, 55.4, 55.1, 51.9, 49.8, 47.9, 42.0, 41.9, 38.1, 38.0, 32.7, 30.7, 30.1, 28.8, 28.3, 28.2, 26.6, 26.4, 26.0, 25.0, 23.7, 22.6, 22.5, 21.6, 17.7, 17.6, 14.5, 9.2. ESI-MS: m / z calculated values, C 71 H 89 N 16 O8 [(M-Cl)+2H] 2+ 647.35; Measured value: 647.63. HRMS (ESI): Calculated m / z value, [C 71 H 89 N 16 O8] + Cl - 1329.6816 [M+H] + ; Measured value: 1329.6809.

[0112] In vitro experiments: Preparation of storage solution: Dissolve NO-AH in MilliQ water to obtain a 2.5 mM stock solution. The enzyme stock solution can be used in its commercial form or dissolved in the appropriate buffer or MilliQ water as indicated by the manufacturer.

[0113] Unless otherwise stated, all enzyme assays were performed in 96-well plates in a TECAN Spark (Mennedorf, Switzerland) multimode microplate reader.

[0114] Absorbance and fluorescence measurementsNO-AH (10 μM) was incubated with and without OmpT (18 μg / mL) and APN (1 mU), and NO-AH (10 μM) was incubated alone with OmpT (18 μg / mL) or APN (1 mU) in PBS (pH 7.4) at 37 °C for 2 hours in a working volume of 200 μL. Afterwards, absorbance scans were performed, and fluorescence measurements were taken at 660 nm excitation. All experiments were performed in triplicate. Selectivity studies: NO-AH (10 μM) was co-incubated with different enzymes in PBS (pH 7.4) at 37°C for 2 hours. The working volume was 200 μL. The enzymes included OmpT (18 μg / mL), APN (1 mU), lysozyme (LZ; 18 μg / mL), β-galactosidase (β-Gal; 1 unit), nitroreductase (NTR; 18 μg / mL), caspase 3 (Casp3; 0.36 mU), cathepsin B (CtSB; 18 μg / mL), and glutathione (GSH; 18 μg / mL). NADH (10 μM) was added as a cofactor to activate NTR. Fluorescence measurements were performed at 660 nm excitation and 720 nm emission. All experiments were performed in triplicate. HPLC analysis: In the presence of both OmpT and APN (1 mU), OmpT was added sequentially followed by APN. Alternatively, OmpT (18 μg / mL) or APN (1 mU) alone was used to incubate vials of NO-AH (10 μM), as well as vials of NO-AH (10 μM), R-CyOH (10 μM), and CyOH (10 μM). These were incubated in PBS (pH 7.4) at 37°C for 2 hours, with a working volume of 200 μL. After incubation, 200 μL of each mixture was injected into a Shimadzu HPLC (acetonitrile / water) instrument to obtain HPLC chromatograms with an absorbance of 660 nm.

[0115] Enzyme kinetics of a single enzyme Different concentrations of NO-AH (2.5 μM, 5 μM, 10 μM, 20 μM, 25 μM) were co-incubated with OmpT (3.6 μg / mL) at 37°C for 5 minutes, with a working volume of 200 μL PBS (pH 7.4). After incubation, the mixture was injected into a Shimadzu HPLC (acetonitrile / water) instrument for quantitative analysis. Different concentrations of R-CyOH (1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) were co-incubated with APN (1 mU) at 37°C for 120 minutes, with a working volume of 200 μL PBS (pH 7.4). Initial reaction rate (μM min)-1 The Michaelis-Menten curve was calculated and fitted based on the substrate concentration.

[0116] The kinetic parameters were calculated using the Michaelis-Menten equation: V = Vmax x [S] (Km + [S]), where V is the initial velocity and [S] is the substrate concentration. The calculated parameters are as follows:

[0117] Microbial culture: All *E. coli* bacterial cells, MRSA, and *Proteus mirabilis* were cultured in Luria-Bertani (LB). *Enterococcus faecalis* was cultured in brain heart infusion broth. First, all bacterial strains were streaked from their glycerol stock onto agar plates and incubated at 37°C for 12–18 hours. Next, single colonies of each bacterial strain were inoculated into 5 mL of broth and incubated overnight at 37°C and 220 rpm. Afterward, bacterial cells were obtained by centrifugation at 4000 g for 10 minutes, washed twice with PBS, and resuspended in 1 mL of PBS. The suspension was diluted accordingly to OD. 600 = 1, to get approximately 10 8 The concentration of CFU / mL was used for subsequent experiments.

[0118] Bacterial imaging: NO-AH (10 μM) was co-incubated with different bacterial strains in PBS (pH 7.4) at 37°C for 2 hours at 220 rpm, with a working volume of 100 μL. After incubation, the bacterial cells were washed twice with PBS and fixed with 15% v / v formaldehyde. 1.5 μL of the bacterial suspension was dropped onto a coverslip and covered with another coverslip for fixation. Then, confocal laser scanning microscopy images were acquired using a Carl Zeiss LSM 800 confocal laser microscope at 640 nm excitation and 700 / 30 nm emission. For experiments involving inhibition, uropathogenic Escherichia coli was inhibited using an OmpT inhibitor (3.2 mM) with the peptide sequence Ac-RffRr, in which the amino acid at the P1' position of the peptide sequence Ac-RffRr is replaced by D-Arg. This peptide sequence has been reported as an inhibitor of OmpT activity (V. Hritonenko, C. Stathopoulos, Mol Membr Biol, 2007, 24, 395). Inhibition was first performed for 1 hour, followed by the addition of NO-AH and incubation at 37°C in PBS (pH 7.4) for another 2 hours. Alternatively, uropathogenic Escherichia coli cells were also first inhibited for 30 minutes with phenbutazone (APN inhibitor; 1 mM), followed by the addition of NO-AH and incubation at 37°C in PBS (pH 7.4) for another 2 hours. All experiments were performed in triplicate.

[0119] Selective recognition of co-cultures of two different bacterial strains: CFT073 and *Proteus mirabilis* were co-incubated with different proportions of NO-AH (10 μM) in PBS (pH 7.4) at 37°C for 2 hours at 200 rpm using a working volume of 100 μL. After incubation, the bacterial cells were washed twice with PBS and fixed with 15% v / v formaldehyde. 1.5 μL of the bacterial suspension was placed on a coverslip and covered with another coverslip for fixation. Confocal laser scanning microscopy images were then acquired using a Carl Zeiss LSM 800 confocal laser microscope at 640 nm excitation and 700 / 30 nm emission. All experiments were performed in triplicate.

[0120] Selective localization of co-cultures of two different bacterial strains: Proteus mirabilis was first stained with Hoechst (10 μM) for 30 minutes at room temperature, followed by a single wash with PBS. Subsequently, the Hoechst-stained Proteus mirabilis was co-cultured with CFT073 and NO-AH (10 μM) in PBS (pH 7.4) at 200 rpm for 30 minutes at 37°C with or without APN in the presence of APN. After incubation, the bacterial cells were washed twice with PBS and fixed with 15% v / v formaldehyde. 1.5 μL of the bacterial suspension was placed on a coverslip and fixed with another coverslip. Confocal laser scanning microscopy images were then acquired using a Carl Zeiss LSM 800 confocal laser.

[0121] Flow cytometry analysis: CFT073 and *Proteus mirabilis* were co-incubated with different proportions of NO-AH (10 μM) in PBS (pH 7.4) at 37°C for 2 hours at 200 rpm using a working volume of 100 μL. After incubation, the bacterial cells were washed twice with PBS and fixed with 15% v / v formaldehyde. Then, 10 μL of each sample was added to 1 mL of PBS and analyzed on a BD Fortessa flow cytometer. Flow cytometry analysis was performed using Flowjo.

[0122] In vivo experiments: In vivo imaging was performed on live mice with a urinary tract bacterial infection model. All animals were cared for and treated according to the IACUC instructions and guidelines (NTU IACUC, protocol number A20065). A single colony of *E. coli* CFT073 was first inoculated into 5 mL of trypsin-soy broth and incubated overnight. Next, 2 mL of the overnight culture was added to another 5 mL of TSB and incubated for 2 hours. Then, 2 mL of the bacterial culture was harvested and resuspended in 1 mL of TSB. OD was measured. 600The value was 1.263. Then, 10 μL of this culture was added to 1 mL of TSB, which was ultimately used to infect mice. Female BALB / c mice were anesthetized accordingly, the urethral orifice was disinfected, and a catheter was inserted into the bladder via the urethra. 50 μL of bacterial culture was injected into the bladder through the catheter. The catheter was removed, and the urethral orifice was clamped using small hemostatic forceps. Mice were infected at different time points: 6 hours, 24 hours, and 48 hours. Each infection time point was characterized by bacterial counts, H&E staining of the bladder, urethra, and kidneys, and measurement of interleukin-6 levels using an ELISA IL-6 kit. For the established urinary tract bacterial infection model, female BALB / c mice were first injected with *E. coli* CFT073 and infected for 6 hours. Then, at different time points after bacterial infection, 50 μL of NO-AH (50 μM) was injected via the bladder or tail vein. NIRF images of live mice were obtained using the IVIS Lumina II imaging system.

[0123] Example 1 - In vitro characterization of NO-AH (compound 7): The stability of NO-AH in buffer solutions was investigated (see [link]). Figure 4 Measurements showed that absorbance and fluorescence intensity were relatively consistent after 24 hours, indicating that NO-AH did not undergo significant degradation.

[0124] The optical properties of NO-AH and its reactivity to the co-enzymatic digestion of OmpT and APN were also examined. As shown in Figure 5, NO-AH (10 μM) itself exhibited two characteristic absorption peaks at 604 nm and 654 nm (Figure 5b), while co-incubation of NO-AH with OmpT and APN resulted in a red shift in absorbance (Figure 5b). Furthermore, a significant fluorescence enhancement (approximately 3-fold) was observed at 720 nm when co-incubated with both enzymes (Figure 5c). As a control, NO-AH showed a minimal absorbance shift when treated with OmpT or APN alone. Figure 6 a). Similarly, less fluorescence enhancement was observed when NO-AH was incubated with the enzyme alone ( Figure 6 b). These optical responses clearly indicate that simultaneous OmpT and APN cleavage is required to provide sufficient fluorescence enhancement for NO-AH.

[0125] Furthermore, the reaction of similar bacterial enzymes to NO-AH was monitored using high-performance liquid chromatography (HPLC). As shown in Figure 5d, when NO-AH (10 μM) was first co-incubated with both OmpT and APN, two peaks appeared: the peak at 9.2 min corresponded to the R-CyOH substrate itself, and the peak at 12.8 min belonged to the CyOH molecule. HPLC analysis also verified the sequential cleavage effect of these two enzymes. As shown in Figure 5d, when NO-AH was first co-incubated with OmpT for 30 min, a new peak with a retention time of 9.2 min was observed, which was the same as the R-CyOH substrate itself, confirming that R-CyOH was generated after OmpT cleavage. Subsequently, APN was added to the same mixture and incubated for another 90 min, and a new peak with a retention time of 12.8 min was observed, belonging to the CyOH molecule, indicating that CyOH was released from R-CyOH after APN protease cleavage. As a control, individual enzymes were co-incubated with the NO-AH probe separately. Figure 7 As shown, incubation of OmpT with NO-AH produced a peak at 9.2 min, indicating enzyme cleavage and R-CyOH formation. However, direct co-incubation of APN with NO-AH failed to hydrolyze the probe molecule into CyOH, and an HPLC peak with a retention time of 9.4 min was observed, close to the retention time of the NO-AH probe itself. These results clearly demonstrate the synergistic activation of NO-AH recognition by the two enzymes. Further analysis of the enzyme kinetics of OmpT for NO-AH and APN for R-CyOH was conducted using HPLC. Within 5 min, when co-incubated with OmpT, the NO-AH molecule was readily cleaved to generate R-CyOH. Calculated kinetic constants showed that K... m = 1.54 μM and K cat = 12.33 min -1 (Figure 5e and) Figure 8 a). On the other hand, within 2 hours, the R-CyOH substrate can be cleaved by APN to release CyOH, resulting in a reasonable kinetic constant K. m = 0.382μM and K cat = 10.24 min -1 (Figure 5f and) Figure 8 b). These results demonstrate that each enzyme, OmpT and APN, exhibits excellent selective recognition of their respective substrates, NO-AH and R-CyOH, in buffer solution.

[0126] Finally, the enzymatic selectivity of NO-AH was investigated by individually screening OmpT, APN, and various commonly used biomolecules and components (see references J. Weng, Y. Wang, Y. Zhang, D. Ye, J. Am. Chem. Soc. 2021, 143, 18294–18304; TC Do, J. Lau, C. Sun, S. Liu, KT Kha, ST Lim, YY Oon, YP Kwan, JJ Ma, Y. Mu, X. Liu, TJ Carney, X. Wang, B. Xing, Sci. Adv. 2022, 8, eabq2216; J. Fang, Y. Zhao, A. Wang, Y. Zhang, C. Cui, S.Ye, Q. Mao, Y. Feng, J. Li, C. Xu, H. Shi, Anal. Chem. 2022, 94). Examples of common biomolecules include lysozyme (Lz), β-galactosidase (β-Gal), nitroreductase (NTR), caspase 3 (Casp3), cathepsin B (CtsB), and glutathione (GSH). As shown in Figure 5g, co-incubation with these common biomolecules showed negligible fluorescence enhancement. Although some nonspecific signals were observed after individual interactions with OmpT or APN, the NO-AH probe of this invention still exhibited excellent selectivity for the sequential activation of bacterial enzymes OmpT and APN.

[0127] Example 2: In vitro imaging of live bacteria using NO-AH (compound 7): 2a) Specific and selective fluorescent bacterial imaging for urinary tract pathogenic Escherichia coli The imaging feasibility of NO-AH's potential performance in different live bacterial cultures was investigated.

[0128] Three types of urinary tract pathogenic Escherichia coli strains with high OmpT expression—Escherichia coli CFT073, Escherichia coli UTI89, and Escherichia coli J96—were selected as target pathogens (see Desloges). et al ., MicrobiologyOpen, 2019, 8, e915). In contrast, methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis (MRSA) were selected. E. faecalis ) and Proteus mirabilis ( P. Mirabilis As controls, these were typically reported as pathogenic species lacking OmpT. After co-incubation with NO-AH (10 μM), live bacterial cultures were examined by confocal microscopy for fluorescence imaging analysis (see reference Wang). et al ., Angew. Chem. Int. Ed. Engl. 2021, 60, 16900-16905). like Figure 9 As shown in a and 9b, strong fluorescence signals were clearly observed in strains CFT073, UTI89, and J96 compared to other strains lacking OmpT.

[0129] As a negative control, the NO-AH probe was also co-incubated with *E. coli* strains (such as BL21, a commonly used *E. coli* strain lacking OmpT expression in its outer membrane). As expected, very low fluorescence was observed in *E. coli* BL21. Figure 10 (a and 10b). These optical images clearly demonstrate the impressive imaging capabilities of NO-AH and its excellent selectivity for UPECs expressing OmpT.

[0130] In addition, we further selected CFT073 and UTI89, which are known typical urinary tract pathogenic Escherichia coli strains that cause UTIs (see the literature Schaale). et al (See *Mucosal Immunol.* 2016, 9, 124-136), enzyme activity was verified by treatment with an OmpT inhibitor (e.g., Ac-RffRr) (see reference Hritonenko). et al ., Mol. Membr. Biol. 2007, 24, 395-406). like Figure 9 As shown in c and 9d, confocal imaging revealed significant fluorescence in CFT073 and UTI89 strains when co-incubated with NO-AH. However, in the presence of the OmpT inhibitor Ac-RffRr, fluorescence was significantly reduced in images of bacteria treated with the NO-AH probe. This further confirms the excellent specificity of the NO-AH probe for OmpT-expressing bacterial strains. Similarly, we also verified the specific enzymatic activity of APN in CFT073 and UTI89 by inhibiting APN. Figure 9 In c and 9e, fluorescence signals were found to be significantly reduced in the presence of APN inhibitors, such as phenbutazone. These results definitively demonstrate the necessity of the synergistic action of the two proteases to specifically activate the NO-AH molecular probe, thereby enhancing fluorescence imaging in urinary tract pathogenic Escherichia coli strains. This enzyme-specific bacterial imaging can be further quantified using fluorescence readouts recognized by CFT073, with observed results as low as approximately 10-1. 4 CFU / mL Figure 11 This is comparable to most previously reported analytical levels (see Mendive-Tapia). et al., Angew. Chem. Int. Ed. Engl. 2022, 61,e202117218; Meile et al ., Nat. Commun. 2023, 14, 4336; van der Zee et al .,PLoS One 2016, 11, e0150755; Pandey et al ., Nat. Chem. 2021, 13, 895-901; Park et al ., Anal. Chem. 2022, 94, 4756-4762; Zhang et al ., Int. J. Nanomed. 2022,17, 3723-3733).

[0131] b) Coenzyme activation of NO-AH for localization imaging in bacterial periplasm: A unique element in our well-designed NO-AH is the synergistic action of bacterial enzymes to specifically target and activate fluorescent probes within the bacteria.

[0132] like Figure 9 As shown in the diagram, co-incubation of NO-AH with the OmpT-positive CFT073 strain resulted in specific bacterial imaging at approximately 700 nm. Interestingly, magnified fluorescence imaging revealed a unique profile formed on the CFT073 surface. Figure 9 f), suggesting the possibility of well-localized fluorescence in the periplasmic region.

[0133] In addition, the specificity and localization imaging capability of NO-AH in mixtures of live bacterial cultures were investigated. Typically, CFT073 was first co-cultured with varying proportions of *Proteus mirabilis* (a morphologically similar but OmpT-free control strain) while maintaining a total bacterial count of approximately 10⁻⁶. 8 CFU / mL. As shown in Figure 12a, when co-incubated with NO-AH (10 μM) and bacterial co-culture, a clear increasing trend in fluorescence signal was observed as the proportion of CFT073 in the bacterial co-culture increased. Similarly, the increased fluorescence signal was quantified using cell counting (FCM) analysis (see reference Widjaja). et al ., Chem. Commun. (Camb.) 2017, 53, 3330-3333; Jaber et al(ACS Cent. Sci. 2020, 6, 1698-1712), and it was observed that the fluorescence intensity increased with the increase of the proportion of CFT073 in the bacterial mixture. These fluorescence response results further confirm that, in mixed bacterial environments, NO-AH has superior imaging selectivity and regional targeting for CFT073 compared to Proteus mirabilis.

[0134] Inspired by these promising findings, we then investigated whether NO-AH could be activated through sequential enzyme activity and its distribution within the bacterial cell walls of UPEC bacteria, thereby enabling bacterial localization imaging. To this end, *Proteus mirabilis* (PM) was first labeled with Hoechst (10 μM), then co-cultured with OmpT-expressing CFT073, followed by co-incubation with NO-AH (10 μM) for imaging analysis (Fig. 12c). The confocal microscopy images in Fig. 12d show a clear bacterial difference between *Proteus mirabilis* (PM) and CFT073, where the blue signal (Hoechst) represents *Proteus mirabilis*, while the red signal (NO-AH) belongs to CFT073, with minimal red staining observed in *Proteus mirabilis* (PM). This response further confirms the selective and periplasmic localization of NO-AH only in CFT073.

[0135] Interestingly, the observation of red fluorescence around CFT073 (Fig. 12d) highlights the importance of the spatial distribution of the two enzymes in capturing fluorescence signals through the sequential activation of OmpT and APN to facilitate bacterial localization imaging in the periphyseal of CFT073.

[0136] Importantly, to further evaluate the necessity of the spatial localization of the two enzymes and their impact on localization imaging, interference experiments were conducted to disrupt the spatial distribution of bacterial enzymes by introducing APN into a co-cultured bacterial mixture.

[0137] The addition of APN (abcam; EC3.4.11.2) to bacterial co-cultures resulted in increased red fluorescent staining in the total bacterial count. Unlike the co-culture study in Figure 12d without added APN, more bacteria (already stained with blue fluorescence) were found to be labeled with red fluorescence (Figure 12e). In this case, it is believed that OmpT on the outer membrane of CFT073 first interacts with NO-AH to produce R-CyOH. Since the added APN is present in the culture medium, these APNs cleave arginine residues from the R-CyOH to provide free CyOH, which infiltrates into the co-cultured bacterial environment. This leads to increased red fluorescent staining, particularly in OmpT-negative Proteus mirabilis (PM), where significant red bacterial labeling was observed.

[0138] Therefore, with the addition of APN to the mixed bacterial environment, the red fluorescence signal could no longer be effectively localized only to CFT073 (Figs. 12d and 12e), and there was more fluorescence signal overlap between the red and blue channels, as evidenced by the increase in the Pearson correlation coefficient value from 0.485 to 0.614. Figure 13 The phenomenon further demonstrates that the added APN can induce the diffusion of free CyOH in the mixed bacterial environment, which eventually stains and enters *Proteus mirabilis* (PM), interfering with the initial localization imaging of UPEC. These results clearly show that the specificity and localization imaging capability of NO-AH are largely influenced by the combined effects of OmpT and APN located in different spaces of the UPEC bacterial cell wall.

[0139] Furthermore, potential interference from the abundant metabolic enzymes present in the complex cellular environment of living systems was considered. Specifically, commonly used proteases (trypsin) were added to live cultures of CFT073 (OmpT(+)) and *Proteus mirabilis* (OmpT(-)), respectively, and then co-incubated with NO-AH (10 μM). Notably, the addition of trypsin to CFT073 showed negligible fluorescence changes compared to co-incubation with NO-AH without trypsin. Figure 14 Furthermore, the fluorescence signal of NO-AH showed a consistent response in the presence of trypsin and its inhibitor (BBI), further confirming that the metabolic enzymes had minimal effect on UPEC staining. As expected, the fluorescence intensity of the *Proteus mirabilis* group with added trypsin was similar to that of the untreated group, both exhibiting weak fluorescence emission. These responses indicate that these metabolic enzymes have minimal interference with the specificity of NO-AH for UPEC imaging expressing OmpT. Figure 14 ).

[0140] Furthermore, throughout the imaging studies, live / dead staining and cell analysis confirmed that NO-AH has excellent biocompatibility. Figure 15 and Figure 16 This suggests that the NO-AH probe has the potential to selectively label CFT073 expressing OmpT and to enable precise in vivo UTI imaging.

[0141] Example 3: In vivo mouse imaging using NO-AH (compound 7): Inspired by the excellent in vitro response of NO-AH, we explored its ability to image bacteria in vivo. A mouse model of urinary tract bacterial infection was established in which Escherichia coli CFT073 was directly injected into the bladder of female BALB / c mice via the urethra, resulting in infection over an extended period.

[0142] After infection, the amount of *E. coli* CFT073 in the urine, bladder, urethra, and kidneys of mice was measured. Figure 17 As shown in Figure a, the highest level of *E. coli* CFT073 was observed 6 hours post-infection and gradually decreased over time through excretion from the body. Simultaneously, immune responses in relevant organs were tested, and significantly elevated interleukin-6 levels were observed 6 hours post-infection, signaling a marked inflammatory response induced by bacterial infection in live mice. Figure 17 b). In addition, H&E staining ( Figure 18 It also showed swelling and shedding of epithelial cells in the bladder and urethral regions, with numerous bacterial spots appearing in the bladder 6 hours after infection. Figure 18 (Circles) indicate that a successful UTI mouse model was established in vivo during the 6-hour bacterial infection period for real-time imaging using NO-AH (Figs. 19a-c). To monitor the performance of the NO-AH probe in vivo and potential fluorescence changes, NO-AH (50 μM) was directly injected into the bladders of live mice with a 6-hour infection model, and the mice were immediately imaged at different time points after NO-AH treatment using the IVISLumina II imaging system. Compared to the same probe injected into healthy mice, a significant increase in fluorescence was observed after intrabladder injection of NO-AH (50 μM) in UTI-infected mice, which was maintained for a period of 3 hours (Figs. 19d-e). This significant observation suggests that a selective response to urinary tract infection by NO-AH is feasible. As a control, the fluorophore CyOH (50 μM) was also injected directly into the bladders of infected or uninfected live mice alone, and the in vivo fluorescence imaging signal remained relatively consistent for both healthy and infected mice. No significant fluorescence differences were observed in healthy and UTI-infected mice (Fig. 19d to e), characterized by non-selective fluorescent staining of CyOH molecules themselves. In contrast, our dual-enzyme-responsive NIR NO-AH probe can specifically respond to the UTI environment and enable real-time regional imaging of urinary tract infections.

[0143] Inspired by this promising in vivo imaging study, we further examined the potential for in vivo UTI imaging within a specific timeframe following intravenous injection of the NO-AH probe (Fig. 19f). As shown in Figs. 19g and 19h, the bladder region of mice exhibited significant fluorescence enhancement, with the fluorescence signal increasing over 3 hours. As a control, healthy mice were given the same intravenous injection of NO-AH, and no fluorescence enhancement was observed during the 3-hour timeframe. Furthermore, 4 hours later, mice were sacrificed after the same intravenous injection of NO-AH to observe fluorescence in the organs. Fig. 19g clearly shows the signal confined in the bladder of infected mice (see dashed circled area). These results demonstrate that NO-AH can be used for real-time imaging to locate and monitor urinary tract infections in live animals.

[0144] It is noteworthy that inaccurate urine testing throughout the entire UTI treatment process in hospitals can easily lead to misdiagnosis of UTI, which increases the risk of recurrence and prolongs treatment time, potentially impacting people's health complications and the economic burden caused by the resulting medical costs. Therefore, we investigated the feasibility of our NIR NO-AH probe sensitively reading out UTI status caused by UPEC strains. As shown in Figure 19i, two days after CFT073 UPEC infection in live mice, no detectable bacteria were found in mouse urine samples, while ex vivo tissue infection analysis showed significant residual amounts of CFT073 (approximately 10 μg / mL) in the bladder and urethral regions. 5 (CFU / g). Meanwhile, IL-6 levels clearly indicated an immune response 2 days after CFT073 infection (Fig. 19j). These observations suggest that bacterial infection still occurred in mice, highlighting the possibility of misdiagnosis of UTI due to inaccuracies in routine urine testing. However, by administering NO-AH to live mice infected with CFT073 for 2 days, in vivo imaging revealed significant fluorescent signals in the bladder and urethra, similar to those observed in mice infected with CFT073 for 6 hours (Fig. 19k). These findings demonstrate that NO-AH can directly reveal the state of UTI in vivo, a readability impossible in typical urine samples (Fig. 19l).

Claims

1. A peptide probe according to formula (I): in, P does not exist or is -C(O)C 1-6 alkyl; Z represents a peptide containing the amino acid sequence according to formula (II): in, n is an integer from 0 to 6; m is an integer from 0 to 5; Each X 1 Independently selected from any amino acid; X 3 For Arg or Lys; Each X 4 Independently selected from any amino acid; When m is 0, X 5 It does not exist or is selected from any amino acid; and When m is between 1 and 5, X 5 Selected from any amino acid; L represents a bond or a self-splitting linker; and Q is a detectable marker; or The peptide probe salt or solvate.

2. The peptide probe according to claim 1, wherein, When each X 1 X 4 and X 5 When present, they are independently selected from any standard amino acid or its D-amino acid.

3. The peptide probe according to claim 1 or 2, wherein: Each X 1 The amino acids are independently selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; X 3 For Arg or Lys; Each X 4 The amino acids are independently selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids; When m is 0, X 5 The D-amino acid present or selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro, or their D-amino acids is absent or selected from them; and When m is between 1 and 5, X 5 Selected from His, Ala, Gly, Ile, Leu, Met, Trp, Phe, Val, Lys, Arg, Pro or their D-amino acids.

4. The peptide probe according to any one of claims 1 to 3, wherein each X 1 X 4 and / or X 5 At least one of them is independently selected from any basic amino acid, for example, at least one X 1 For His, Lys, Arg, or their D-amino acids, at least one X 4 His, Lys, Arg, or their D-amino acids, and / or X 5 It is His, Lys, Arg, or their D-amino acids.

5. The peptide probe according to any one of the preceding claims, wherein at least one X 1 For D-Phe and / or at least one X 4 It is D-Phe.

6. The peptide probe according to any one of the preceding claims, wherein Z represents a peptide consisting of an amino acid sequence according to formula (IIa): in, X 1 Selected from His, Lys, Arg, or their D-amino acids, X 2 ′ and X 3 Each is independently either Phe or D-Phe, and X 5 ′ is either Arg or Lys; and Where L is X 5 The key connected to Q, or L is connected to X. 5 ′ is connected to the self-split linker of Q.

7. The peptide probe according to claim 6, wherein X 1 ′ is Arg, and X 2 ′ and X 3 Each is D-Phe.

8. The peptide probe according to any one of the preceding claims, wherein P is -C(O)C 1-4 Alkyl groups, for example, P is an acetyl group.

9. The peptide probe according to any one of the preceding claims, wherein the peptide probe is as shown in SEQ ID NO: 1: in, L represents a bond or a self-splitting linker; and Q is a detectable marker; or The peptide probe salt or solvate.

10. The peptide probe according to any one of the preceding claims, wherein Q is selected from the group consisting of coumarin, naphthaleneimide, xanthene, rhodamine, anthocyanin, porphyrin, nitrobenzofuran, and boron-dipyrrolemethylene.

11. The peptide probe according to any one of the preceding claims, wherein Q is a near-infrared (NIR) fluorescent dye, for example, Q may be selected from the group consisting of CyOH, Cy5.5 and Cy7.

12. The peptide probe according to any one of claims 1 to 9, wherein Q is a chemiluminescent label or a bioluminescent label.

13. The peptide probe of claim 12, wherein Q is selected from the group consisting of luminol, fluorescein, chemiluminescent labeling based on 1,2-dioxane, and acridine ester.

14. The peptide probe according to any one of the preceding claims, wherein L is a linker according to formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg) or (Lh): in, Each R a Independently select from the groups composed of O and NH. Each R b Independently selected from the groups consisting of O, NH, -OC(O)O-, and -OC(O)NH-; and This indicates the binding site between the connecting portion and Z, while This indicates the binding site between the connecting portion and Q.

15. The peptide probe according to any one of the preceding claims, wherein L is a linker according to the following formula: ， in, This indicates the binding site between the connecting portion and Z, while This indicates the binding site between the connecting portion and Q.

16. The peptide probe according to claim 1, wherein the peptide probe is as shown in SEQ ID NO: 3: SEQ ID NO:

3.

17. The peptide probe of claim 16, wherein the peptide probe is as shown in SEQ ID NO: 4: SEQ ID NO:

4.

18. A pharmaceutical composition comprising a peptide probe according to any one of the preceding claims and a pharmaceutically acceptable carrier.

19. A method for detecting a urinary tract infection in a urine sample obtained from a subject, the method comprising: a) Provide a urine sample obtained from the subject; b) Contact the urine sample with the peptide probe according to any one of claims 1 to 17; c) Determine the presence of urinary tract pathogenic Escherichia coli in the urine sample by detecting the detectable markers of the peptide probe in the urine sample. E. coli ).

20. A method for determining one or more suitable therapeutic agents for treating a urinary tract infection in a subject, the method comprising: i) Provide a urine sample obtained from the subject; ii) Contact the urine sample with the peptide probe according to any one of claims 1 to 17; iii) Determine the presence of urinary tract pathogenic Escherichia coli in the urine sample by detecting the detectable marker of the peptide probe in the urine sample; as well as iv) Based on the detection of the detectable label of the peptide probe, determine one or more suitable therapeutic agents for treating the subject's urinary tract infection.

21. A method for treating a subject with a urinary tract infection, the method comprising: A. Provide a urine sample obtained from the subject; B. Contact the urine sample with the peptide probe according to any one of claims 1 to 17; C. Determine the presence of urinary tract pathogenic Escherichia coli in the urine sample by detecting the detectable markers of the peptide probe in the urine sample; as well as D. Based on the detection of the detectable label of the peptide probe in the urine sample, administer a dose of one or more therapeutic agents for treating urinary tract infections to the subject.

22. A method for determining one or more suitable therapeutic agents for treating a urinary tract infection in a subject, the method comprising administering to the subject a peptide probe according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, and determining the one or more suitable therapeutic agents for treating the urinary tract infection by detecting a detectable marker of the peptide probe in the subject's bladder, urethra, and / or urine.

23. A method for treating a patient with a urinary tract infection, the method comprising administering to a subject a peptide probe according to any one of claims 1 to 17, or a pharmaceutical composition according to claim 18, detecting a detectable marker of the peptide probe in the subject's bladder, urethra, and / or urine, and administering to the subject a dose of one or more therapeutic agents for treating the urinary tract infection.

24. The method according to any one of claims 20 to 23, wherein the one or more therapeutic agents are antibiotics, such as antibiotics selected from the group consisting of trimethoprim, sulfamethoxazole, fosfomycin, nitrofurantoin, cephalexin, ceftriaxone, cefaclor, tetracycline and antimicrobial peptides.

25. The method according to any one of claims 22 to 24, wherein the peptide probe is administered to the subject by urethral injection or intravenous injection.

26. The method according to any one of claims 22 to 25, wherein the urinary tract infection of the subject is caused by urinary tract pathogenic Escherichia coli.

27. An in vitro diagnostic kit for detecting, screening, monitoring, classifying, selecting treatment for urinary tract infections in subjects, determining whether treatment is effective in subjects with urinary tract infections, and / or predicting urinary tract infections in subjects, said in vitro diagnostic kit comprising one or more peptide probes according to any one of claims 1 to 17.

28. The in vitro diagnostic kit of claim 27, wherein the kit comprises a peptide probe according to any one of claims 1 to 17, wherein Q is a luciferin, and the in vitro diagnostic kit further comprises a luciferase.

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