Peptide probes

Abstract

The present invention provides a peptide probe according to formula (I) as defined herein The peptide probe of the present invention finds utility in the detection, screening, monitoring, classification, selecting treatment for, ascertaining whether treatment is working in, and / or prognostication of a urinary tract infection. The present invention also provides compositions comprising the peptide probe of the invention, methods of using the peptide probes in the detection and treatment of a urinary tract infection, and an in vitro diagnostic kit comprising the peptide probe of the invention.

Description

[0001] PEPTIDE PROBES

[0002] FIELD OF INVENTION

[0003] The present invention relates to peptide probes that find utility in the detection, screening, monitoring, classification, selecting treatment for, ascertaining whether treatment is working in, and / or prognostication of a urinary tract infection. The present invention also provides compositions comprising the peptide probe of the invention, methods of using the peptide probes in the detection and / or treatment of a urinary tract infection, and an in vitro diagnostic kit comprising the peptide probe of the invention.

[0004] BACKGROUND

[0005] The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0006] Escherichia coli (E. coli) is one of the world’s most common food and water-borne pathogenic bacteria infecting millions worldwide with adverse conditions such as urinary tract infections, diarrhoea, blood infections and respiratory illnesses (WHO, WHO Estimates of the Global Burden of Foodborne Diseases, World Health Organization, 2015). However, despite the imminent situation, the current standard method for detection is still via conventional bacteria isolation and culture which could be extremely time consuming and labour 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 persistent cases of infection, antibiotics are prescribed to alleviate the situation which inadvertently results in the surge of undesired antibiotic resistance (Bartoletti et al., European Urology Supplements 2016, 15, SI- 87, and EAU, EAU Guidelines on Urological Infections, European Association of Urology, 2022). Due to the rising annual fatality caused by ingesting E. coli contaminated food and water, several groups have recently reported functional assays to rapidly detect the presence of E. coli in food and water samples. These methods however are still primarily limited to ex vivo detection kits, which greatly help in prior detection to prevent subsequent consumption 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., Angew Chem Int Ed Engl 2020, 59, 18068-18077). However, instead of mere prevention strategies, it is more important to fully study and understand the underlying biological mechanisms associated with the worldwide lethality of E. coli infections in vivo. Unfortunately, such studies still remain elusive to date. As part of commensal intestinal flora, it is not surprising that E. coli usually lacks virulence in the human gastrointestinal tract. Even so, they can still cause intestinal illnesses upon ingestion of pathogenic strains with distinct virulent factors and innate abilities in overcoming host defenses (Mueller eta / ., Treasure Island (FL): StatPearls Publishing, 2023). However, all of these effects can be largely mitigated with a highly regulated safety standard. On the other hand, extraintestinal illnesses caused by E. coli usually results from translocation of commensal gut bacteria into other parts of the body, causing life-threatening conditions such as pneumonia (lungs), bacteremia (blood), and meningitis (brain), among others (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 urinary tract in particular, is the most common extraintestinal site of infection caused by E. coli ascending the urethra through trace transfer of fecal excretions from the intestines (Zagaglia et al., Microorganisms 2022, 10). As such, urinary tract infections (UTIs) are amongst the most common diseases encountered in clinical practice worldwide, affecting 150 million annually with considerable morbidity 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 rate of resistance relative to the antibiotics recommended by current guidelines for the treatment of UTIs and an increasing number of multidrug resistant uropathogenic E. coli (UPEC) isolates were observed (Bruxvoort, et al., Clin Infect Dis 2020, 71 , 100-108, and Mazzariol etal., J Chemother 2017, 29, 2-9), thus prompting the dire need for not only new therapeutic methods, but also a deeper biological understanding to ameliorate the current situation.

[0007] Conventional approaches to identify bacteria includes culture and colony counting that could be labor intensive, time consuming and usually require a series of intricate analysis before reliable results could be obtained (Velusamy et al., Biotechnol Adv 2010, 28, 232-254). As such, these conventional methods limit their applications in bacterial detections. Over the years, various techniques have been emerging to overcome such limitations towards detecting bacteria such as polymerase chain reaction (Lazcka et al., Biosens Bioelectron 2007, 22, 1205-1217), electrochemical sensing (Su et al., Biosens Bioelectron 2011 , 26, 1788-1799), immunology-based (Swaminathan et al., Annual Reviews Microbiology 1994, 48, 401-426), mass spectrometry and chromatography (Zhu etal., Chem Sci 2016, 7, 2987-2995). However, these methods require the use of complicated equipment and involves complex procedures to operate them. Furthermore, in the clinical aspect, the traditional ways for the detection of bacterial infection mainly relies on blood assays such as procalcitonin test (PCT) and C- reactive protein (CRP), and / or nuclear imaging like 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). Nonetheless, these procedures are invasive and inefficient, and potentially provide false positive results that would delay the whole diagnosis process (Yoon etal., Chem 2021 , 9, 743923).

[0008] Therefore, there exists a need for improved or alternative approaches for bacterial detection, in particular uropathogenic E. coll, and preferably with high specificity and sensitivity and without the need of complex machinery and invasive procedures.

[0009] SUMMARY OF INVENTION

[0010] The present invention provides a peptide probe according to formula (I):

[0011] P-Z-L-Q

[0012] (I) wherein,

[0013] P is absent or -C(O)Ci-6alkyl;

[0014] Z represents a peptide comprising an amino acid sequence according to formula (II): [X1]nRX3[X4]mX6

[0015] (II) wherein, n is an integer from 0 to 6; m is an integer from 0 to 5; each X1is independently selected from any amino acid;

[0016] X3is Arg or Lys; each X4is independently selected from any amino acid; when m is 0, X5is absent or selected from any amino acid; and when m is from 1 to 5, X5is selected from any amino acid;

[0017] L is a bond or a self-immolative linker; and

[0018] Q is a detectable label; or a salt or solvate thereof.

[0019] In certain preferred embodiments, in formula (II), when present, each X1, X4, and X5are independently selected from any canonical amino acid or a D-amino acid thereof. For example, in formula (II), each X1may be independently selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; each X4may be independently selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; and when m is 0, X5may be absent or selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; and when m is from 1 to 5, X5may be selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof.

[0020] The present invention also provides a composition comprising a peptide probe of the present invention and a carrier, in certain embodiments, the composition is a pharmaceutical composition comprising a peptide probe of the present invention and a pharmaceutically acceptable carrier.

[0021] The present invention also provides a method of detecting a urinary tract infection in a urine sample obtained from a subject, and a method of determining one or more suitable therapeutic agents for the treatment of a urinary tract infection in a subject. Also disclosed herein is a method of determining one or more suitable therapeutic agents for the treatment of a urinary tract infection in a subject, and a method of treating a urinary tract infection in a subject. Also provided herein is an in vitro diagnostic kit for use in the detection, screening, monitoring, classification, selecting treatment for, ascertaining whether treatment is working in, and / or prognostication of a urinary tract infection in a subject, said in vitro diagnostic kit comprising one or more peptide probes of the present invention.

[0022] Preferred but optional features are set out in the dependent claims. Additional 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. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment.

[0023] BRIEF DESCRIPTION OF DRAWINGS

[0024] FIG. 1 is an illustration of the synergistic activation of NO-AH (Compound 7) by OmpT and aminopeptidase N (APN) in the presence of a urinary tract bacterial infection.

[0025] FIG. 2 shows the synthetic scheme of R-CyOH (a) K2CO3, CH3CN, 50 °C, 6 h; (b) N- ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline (EEDQ), anhydrous dichloromethane (DCM), room temperature (r.t.), 16 h; (c) 1. PBrs, anhydrous tetra hydrofuran (THF), 0 °C, 2 h; 2. N,N- Diisopropylethylamine (DIPEA), 55 °C, 6 h; (d) 1. DCM, triisopropyl silane (TIPS), trifluoroacetic acid (TFA), 0 °C; 2. 5% piperidine in dimethylformamide (DMF), r.t. FIG. 3 shows the synthetic scheme of NO-AH (a) K2CO3, CH3CN, 50 °C, 6 h; (b) EEDQ, anhydrous DCM, r.t, 16 h; (c) 1. PBr3, anhydrous THF, 0 °C, 2 h; 2. K2CO3, 50 °C, 4 h; (d) DCM, TIPS, TFA, 0 °C.

[0026] FIG. 4 shows the in vitro stability of NO-AH in phosphate buffer saline (PBS), (a) Absorbance at 660 nm of NO-AH (10 pM) at 37 °C in PBS upon incubation for 0 h, 2 h, 18 h, and 24 h. (b) Fluorescence emission at 720 nm of NO-AH (10 pM) at 37 °C in PBS upon incubation for 0 h, 2h, 18h, and 24h. Data presented as means ± SD (n=3).

[0027] FIG. 5 shows the in vitro characterization of NO-AH. (a) Schematic visualization of the enzymatic cleavage of NO-AH by OmpT and APN. Enzymatic cleavage of the peptide probe by OmpT results in the release of a peptide with the sequence RFFR (SEQ ID NO: 5). (b-c) Absorbance and fluorescence spectra of NO-AH (10 pM) in the presence and absence of both OmpT and APN for 2 h at 37 °C in PBS (pH 7.4). ( A ex: 660 nm) (d) HPLC analysis of OmpT and APN with NO-AH incubated for 2 h at 37 °C in PBS (pH 7.4). (e) Enzyme kinetics of OmpT with NO-AH (10 pM). (f) Enzyme kinetics of APN with R-CyOH (10 pM). (g) The selectivity analysis towards enzymatic NO-AH reaction (10 pM) after incubation with indicated biomolecules for 2 h at 37 °C in PBS. ( T ex: 660 nm; T ern: 720 nm). (F represents the Fluorescence emission of indicated biomolecules at T ern: 720 nm; Forepresents the baseline fluorescence of NO-AH at T ern: 720 nm). Data presented as means ±SD (n=3).

[0028] FIG. 6 shows (a) absorption spectra and (b) fluorescence spectra of NO-AH (10 pM) in the presence of individual enzymes (OmpT or APN) and both enzymes for 2 h at 37 °C in PBS (pH 7.4).

[0029] FIG. 7 shows the HPLC profile of individual enzymes (OmpT or APN) incubated with NO-AH (10 pM) for 2 h at 37 °C in PBS (pH 7.4).

[0030] FIG. 8 shows (a) Time-dependent HPLC spectra of the formation of R-CyOH under the proteolysis of OmpT with NO-AH (10 pM) at different time points at 37 °C in PBS (pH 7.4). (b) Time-dependent HPLC spectra of the formation of CyOH under the proteolysis of OmpT with R-CyOH (10 pM) at various time points at 37 °C in PBS (pH 7.4).

[0031] FIG. 9 shows in vitro fluorescence bacteria (~108CFU / mL) confocal microscopy analysis of various bacteria strains after incubation with NO-AH (10 pM) with and without OmpT or APN inhibitors, (a-b) Confocal images of various bacteria strains including CFT073, UTI89, J96, MRSA, E. faecalis and P. mirabilis incubated individually with NO-AH (10 pM) in PBS at 37 °C for 2 h, with their fluorescence intensities indicated. ( T ex: 640 nm, T ern: 700 / 30 nm) Scale bar: 2.5 pm. (c-e) Confocal images of uropathogenic E. coli bacteria incubated with NO-AH (10 pM) in the presence and absence of an OmpT inhibitor (Ac-RffRr), and in the presence and absence of an APN inhibitor (Bestatin); CFT073 and UTI89 were incubated with and without the OmpT inhibitor (3.2 mM) for 1 h or APN inhibitor (1.0 mM) for 30 mins first, and then incubate for another 2 h with NO-AH (10 pM) in PBS at 37 °C. ( l ex: 640 nm, l em: 700 / 30 nm) Scale bar: 2.5 pm. (f) Confocal images of CFT073 with NO-AH (10 pM) at scale bar of 10 pm and 5 pm. Data presented as means ±SD (n=3).

[0032] FIG. 10 shows the in vitro fluorescence bacteria (~ 108CFU / mL) confocal microscopy analysis of various E. coli strains after incubation with NO-AH (10 pM). (a-b) Confocal images of CFT073, UT89, J96 and BL21 incubated individually with NO-AH (10 pM) in PBS at 37 °C for 2 h; and their corresponding relative fluorescence intensities, ( ex: 640 nm, l em: 700 / 30 nm.) Scale bar: 2.5 pm. Data presented as means ± SD (n=3).

[0033] FIG. 11 shows the normalised fluorescence intensity of NO-AH (10 pM) upon incubation in with E. coli CFT073 ranging from 104to 10® CFU / mL (n = 3). The dashed line is presented as the mean intensity of normalized fluorescence from NO-AH without bacteria plus three times the standard deviation (SD) of the signal.

[0034] FIG. 12 shows the in vitro fluorescence confocal microscopy analysis of the differentiation between live cultures of CFT073 (~10® CFU / mL) and P. mirabilis (~10® CFU / mL). (a) Confocal images of varying ratios co-culture of P. mirabilis and CFT073 after incubation with NO-AH (10 pM) for 2 h in PBS at 37 °C; Scale bar: 2.5 pm. (b) FCM analysis of varying ratios of coculture of P. mirabilis and CFT073 after incubation with NO-AH (10 pM) for 2 h in PBS at 37 °C. (c) Schematic illustration for the investigation of the spatial distribution of dual enzymes in mixed bacteria, (d) Confocal images of co-culture of Hoechst-stained P. mirabilis and CFT073 after incubation with NO-AH (10 pM) for 30 minutes in PBS at 37 °C. (e) Confocal images of co-culture of Hoechst-stained P. mirabilis and CFT073, with addition of APN into mixed bacterial environment, and then incubation with NO-AH (10 pM) for 30 minutes in PBS at 37 °C (Hoechst: l ex: 405 nm, em: 460 / 30 nm; NO-AH: l ex: 640 nm, l em: 700 / 30 nm) Scale bar: 10 pm and 5 pm. (n=3).

[0035] FIG. 13 shows a scatter plot of the co-localisation of the red and blue pixel intensities from NO-AH and Hoechst respectively, (a) Co-culture of Hoechst-stained P. mirabilis and CFT073 after incubation with NO-AH (10 pM) for 30 minutes in PBS at 37 °C. (b) Co-culture of Hoechst- stained P. mirabilis and CFT073 with addition of APN into mixed bacterial environment, followed by incubation with NO-AH (10 pM) for 30 minutes in PBS at 37 °C. FIG. 14 shows confocal images of CFT073 incubated with NO-AH (10 pM) and Trypsin (1 pg / mL), in the presence and absence of BBI (10 pg / mL), and P. mirabilis incubated with NOAH and Trypsin in PBS (pH 7.4) at 37 °C for 30 minutes. ( A ex: 640 nm, l em: 700 / 30 nm.) Scale bar: 2.5 pm.

[0036] FIG. 15 shows the LIVE / DEAD bacterial viability of CFT073 with NO-AH (10 pM). (a) Fluorescent confocal images of SYTO 9 and propidium iodide (PI) double stained on CFT073 after incubation with NO-AH (10 pM) in PBS at 37 °C for 2 h at emission of 500 nm (green channel) and 635 nm (red channel); (b) and their corresponding fluorescence intensities. 1 ex: 488 nm. A em: 490-520 nm / 600-650 nm. Scale bar: 10 pm. SYTO 9 has high affinity with DNA and generate a green fluorescence for live bacteria, while PI indicates the membrane integrity to generate a red fluorescence for dead bacteria. Data presented as means ± SD (n=3).

[0037] FIG. 16 shows the cell viability of 3T3 / NIH cells after 24 h incubation with varying concentrations of NO-AH. Data presented as means ±SD (n=3).

[0038] FIG. 17 shows the establishment of UTI mouse model, (a) Bacterial count of E. coli CFT073 in urine, bladder, urethra and kidney of the mice after infected with E. coli CFT073 for 6 h, 24 h and 48 h; (b) IL-6 levels in the kidney and bladder and urethra, after infected with CFT073 for 6 h, 24 h and 48 h. Data presented as means ± SD (n=3).

[0039] FIG. 18 shows hematoxylin and eosin (H&E) staining of the bladder, urethra and kidneys of the mice after infected with E. coli CFT073 for 6 h, 24 h and 48 h. Scale bar: 100 pm or 10 pm.

[0040] FIG. 19 shows the in vivo near-infrared fluorescence (NIRF) imaging of living mice, (a) Schematic illustration of the NIRF imaging of UTI infected mice with in-bladder injection of NOAH (50 pM) and imaged over different time points, (b-c) H&E staining and IL-6 levels of the bladder and urethra in the healthy and UTI infected mouse after 6 hours of infection, (d-e) NIRF images for UTI infected mice and healthy mice after in-bladder injection of NO-AH (50 pM) and CyOH (50 pM) separately, and their corresponding fluorescence signals, l ex: 660 nm, l em: 710 nm. (f) Illustration of the NIRF imaging of UTI infected mice with i.v. injection of NO-AH (50 pM) and imaged over different time points, (g-h) NIRF images for UTI infected mice and healthy mice with i.v. injection of NO-AH (50 pM); and their corresponding fluorescence signals, l ex: 660 nm, 1 em: 710 nm, in (g) the fluorescence area of the infected mouse at 4h is highlighted by a dashed circle, (i) Bacterial count of CFT073 present in the urine, bladder and urethra after 2 days of bacterial infection. (j) IL-6 levels of healthy and recurrent UTI mice after 2 days bacterial infection (k) NIRF imaging for 6 h and 2 days UTI infected mice with NO-AH injection (50 pM). l ex: 660 nm, l em: 710 nm (I) Scheme of detecting UTI in vivo. Data presented as means ±SD (n=3).

[0041] DESCRIPTION

[0042] As discussed in more detail below, the present inventors have developed a peptide probe according to formula (I) that utilizes the proteolytic activity of OmpT, an E. coli outer membrane protease, and aminopeptidase N (APN) which is located within the periplasm of E. coli (see FIG. 1). The design of the peptide probe of the invention allows for specific targeting of uropathogenic E. coli through its collaborative response to OmpT and APN, which enables detection of urinary tract infections both in vitro and in vivo. In particular, as disclosed in the Examples herein, the present inventors demonstrate that a peptide probe of the present invention referred to as NO-AH displays great selectivity towards bacteria strains that express OmpT and APN. Furthermore, the specificity of OmpT for NO-AH resulted in low non-specific background fluorescence signals in the cellular microenvironment, which contributed to a sensitive signal readout. In addition, the present inventors demonstrate that NO-AH enabled real-time in vivo visualisation and monitoring of a urinary tract bacterial infection.

[0043] Thus, the present invention provides a peptide probe according to formula (I):

[0044] P-Z-L-Q

[0045] (I) wherein,

[0046] P is absent or -C(O)Ci-6alkyl;

[0047] Z represents a peptide comprising an amino acid sequence according to formula (II):

[0048] [X1]nRX3[X4]mX5

[0049] (II) wherein, n is an integer from 0 to 6; m is an integer from 0 to 5; each X1is independently selected from any amino acid;

[0050] X3is Arg or Lys; each X4is independently selected from any amino acid; when m is 0, X5is absent or selected from any amino acid; and when m is from 1 to 5, X6is selected from any amino acid;

[0051] L is a bond or a self-immolative linker; and

[0052] Q is a detectable label; or a salt or solvate thereof.

[0053] For the avoidance of doubt, in formula (II), n may be any integer selected from 0, 1, 2, 3, 4, 5 and 6; and m may be any integer selected from 0, 1 , 2, 3, 4, and 5. Also, for the avoidance of doubt, in embodiments wherein m is 0 and X5is absent, L is a bond that links X3to Q, or L is a self-immolative linker that links X3to Q; and in embodiments wherein m is from 1 to 5 and X5is selected from any amino acid, L is a bond that links X5to Q, or L is a self-immolative linker that links X5to Q.

[0054] The amino acid sequences disclosed herein are shown with the N-terminus to the left, and where sequences are set out across multiple lines, the N-terminus is to the top left. Within a polypeptide chain, the amino acids are linked by peptide bonds between the carboxyl group of one amino acid and the amine group of the next amino acid in the chain. An individual amino acid is called a “residue” or “amino acid residue” once it is linked in a polypeptide chain. The amino acid sequences listed in the application are shown using standard letter abbreviations for amino acids. In the context of the present disclosure, the term “amino acid” encompasses any naturally occurring amino acid or unnatural amino acid. It includes an amino acid in any chiral configuration. The amino acid may, especially, be a naturally occurring alpha-amino acid. The amino acid may, especially, be a naturally occurring L-amino acid. The amino acid may, especially, be a naturally occurring alpha L-amino acid.

[0055] In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components / features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of’ or the phrase “consists essentially of’ or synonyms thereof and vice versa. Thus, for the avoidance of doubt, in certain embodiments, Z in formula (I) represents a peptide comprising or consisting of an amino acid sequence according to formula (II), as defined herein.

[0056] It should be understood herein that provided the peptide probe of the invention comprises the OmpT proteolytic site RJ.(R / K) (i.e. represented by -RX3- in formula (II)), the flanking amino acids (represented by X1, X4and X5in formula (II)) may be present (and any amino acid) or absent. The flanking amino acids can impart additional beneficial functions to the peptide probe such as improved stability and / or selectivity, but are not necessarily required for the core function of the peptide probe to be realized, i.e. to detect uropathogenic E. coli. In the peptide probe of formula (I), when present, each X1, X4and X5in formula (II) are independently selected from any amino acid. For example, when present, each X1, X4and X5may be a canonical amino acid or a non-canonical amino acid. The term “canonical amino acids” refers to the 20 amino acids that are encoded by the universal genetic code and are the building blocks of most proteins and peptides in organism. The 20 canonical amino acids are alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gin), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (lie), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Vai). For the avoidance of doubt, the canonical amino acids are L -amino acids. The term “non-canonical amino acids” refers to amino acids that are not part of the list of 20 amino acids considered to be canonical amino acids. Examples of non-canonical amino acids include, but are not limited to, ornithine (Orn), citrulline (Cit), 2,4- diaminobutyric acid (DABA), 3-azido alanine, 6-azido lysine and propargylglycine. Further examples of non-canonical amino acids include the corresponding D-amino acid of a canonical amino acids, for example, D-alanine (D-Ala) or D-phenylalanine (D-Phe). In certain embodiments, in formula (II), when present, each X1, X4, and X5is independently selected from any canonical amino acid or a D-amino acid thereof.

[0057] In certain preferred embodiments, in formula (II), each X1is independently selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; X3is Arg or Lys; each X4is independently selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; when m is 0, X5is absent or selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; and when m is from 1 to 5, X5is selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof.

[0058] Including one or more basic amino acids in the peptide portion of the peptide probe of the invention (i.e. Z in formula (I)) can impart a positive charge on the peptide probe when it is in a physiological environment (e.g. an environment with a pH of about 7.4), or a basic environment (e.g. pH 7-9) which may exist within the bladder or urethra of a subject suffering from a urinary tract infection. Without wishing to be bound by theory, the present inventors believe when the peptide probe of the invention displays a net positive charge, the peptide probe will interact and bind to lipids and lipopolysaccharide (LPS) molecules present in the outer membrane of Gram-negative bacteria such as E. coli. Thus, in certain embodiments, in formula (II), at least one of each X1, X4, and / or X5are independently selected from any basic amino acid, for example, independently selected from His, Arg, Lys, Orn, Cit, and a D-amino acid thereof (e.g. Arg or Lys). Typically, in formula (II), at least one X1is His, Lys, Arg or a D- amino acid thereof, at least one X4is His, Lys, Arg or a D-amino acid thereof, and / or X5is His, Lys, Arg or a D-amino acid thereof.

[0059] In certain embodiments, the peptide portion of the peptide probe may include one or non-polar amino acids such Gly, Ala or Vai, or amino acid sequences commonly used as spacer moieties, such as repeating combinations of Gly and Ser. In certain other embodiments, the peptide portion of the peptide probe may include one or more proline, or D-amino acids such as D- Phe. Including one or more proline or D-amino acid in the peptide portion of the peptide probe may improve the peptide probe’s stability to non-specific protease digestion, thus improving the selectivity of the peptide probe. Thus, in certain embodiments, in formula (II), at least one X1, at least one X4, and / or at least one X5is Ala, Gly, He, Leu, Met, Trp, Phe, Vai, Pro or a D- amino acid thereof. For example, in formula (II), at least one X1is D-Phe or at least one X4is D-Phe; or at least one X1is D-Phe and at least one X4is D-Phe. Typically, in formula (II), at least one X1is D-Phe.

[0060] In certain preferred embodiments, in formula (I), Z represents a peptide consisting of an amino acid sequence according to formula (Ila):

[0061] X1'X2'X3'RX5'

[0062] (Ha) wherein,

[0063] X1' is selected from His, Lys, Arg, or a D-amino acid thereof, X2' and X3' are each independently Phe or D-Phe, and X5' is Arg or Lys; and wherein, L is a bond that links X5'to Q, or L is a self-immolative linker that links X5' to Q.

[0064] In certain embodiments, wherein Z in the peptide probe of the present invention represents a peptide comprising (or consisting of) an amino acid sequence according to formula (Ila), X1' is Arg, and X2' and X3' are each D-Phe.

[0065] Including a -C(O)Ci-4alkyl group at the N-terminus of the peptide probe of the present invention may improve its stability to non-specific protease digestion, thus improving the selectivity of the peptide probe. Thus, in certain embodiments, in formula (I), P is -C(O)Ci-4alkyl, for example P is acetyl.

[0066] In certain preferred embodiments, the peptide probe of the present invention is according to SEQ ID NO: 1 :

[0067]

[0068] SEQ ID NO: 1 wherein,

[0069] L is a bond or a self-immolative linker; and Q is a detectable label; or a salt or solvate thereof.

[0070] For example, the peptide probe of the present invention may be according to SEQ ID NO: 2:

[0071] SEQ ID NO: 2 wherein,

[0072] L is a bond or a self-immolative linker; and Q is a detectable label; or a salt or solvate thereof.

[0073] For the avoidance of doubt, in SEQ ID NOs: 1 and 2, L and Q may be according to L and Q as defined herein for formula (I).

[0074] The term “self-immolative linker’’ as used herein refers to a linking moiety that undergoes degradation under specific conditions, in particular, following cleavage of a peptide bond adjacent to the linking moiety. Degradation of the linker results in release of the detectable label of the peptide probe. Degradation of the linking moiety may occur, for example, via an electronic cascade or via an elimination pathway. A well-known example of a self-immolative linker is the aminobenzyl alcohol group (PAB). In certain embodiments of the present invention, in formula (I) (and likewise in SEQ ID NOs: 1 and 2), L may be a linking moiety according to formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg) or (Lh): wherein, each Rais independently selected from the group consisting of O and NH, each Rbis independently selected from the group consisting of O, NH, -OC(O)O- and -OC(O)NH-; and

[0075] A iV indicates the site of attachment of the linking moiety to Z and * indicates the site of attachment of the linking moiety to Q.

[0076] In certain exemplary embodiments, L is a linking moiety according to: wherein,

[0077] ''Vw indicates the site of attachment of the linking moiety to Z and * indicates the site of attachment of the linking moiety to Q; such a linking moiety may be also be referred to herein as a p-aminobenzyl (PAB) linking moiety.

[0078] The term “detectable label” as used herein refers to a molecular label that is attached to the peptide probe that may be detected when attached to the peptide probe, or detected once the detectable label is released from the peptide probe following cleave by the protease OmpT and APN. It should be understood that the present invention is not necessarily limited to the use of any specific type of “detectable label”, provided the detectable label can be attached to the C-terminus of a peptide, directly or via a self-immolative linker, and can be detected once released from the peptide probe. Detection of the detectable label may be carried out by any suitable method, including fluorescence spectroscopy, any other optical methods, or methods such as photoacoustic (PA) imaging. When the detectable linker comprises a functional group that allows direct coupling to the C-terminus of a peptide (e.g. a NH2or OH group), the peptide probe may not comprise a self-immolative linker. Rather, the detectable linker may be directly linked to Z of the peptide probe. Thus, in certain embodiments, in formula (I), when Q is a detectable linker that comprises a functional group that allows direct coupling to the C- terminus of a peptide (e.g. a NH2or OH group), L is a bond.

[0079] In certain embodiments, in formula (I) (and likewise in SEQ ID NOs: 1 and 2), Q may be a fluorescence dye. For example, Q may be is selected from the group consisting of a coumarin, a naphthalimide, a xanthene (e.g. fluorescein, and analogies thereof), a rhodamine, a cyanine, a porphyrin, a nitrobenzofurazan, a nitro be nzofurazan, and a boron-dipyrromethene. In certain embodiments, Q may be a near-infrared (NIR) fluorescence dye. Thus, in certain preferred embodiments, Q may be selected from the group consisting of CyOH, Cy5.5, and Cy7. In certain other embodiments, in formula (I) (and likewise in SEQ ID NOs: 1 and 2), Q may be a chemiluminescence label or a bio-luminescence label. For example, Q may be selected from the group consisting of luminol, luciferin, a 1 ,2-dioxetane-based chemiluminescent label, and an acridinium ester. The use of chemiluminescence label or a bio-luminescence label may be preferred in the clinic as it may allow detection of the detectable label by eye without the need of specialist equipment.

[0080] For the avoidance of doubt, other detectable label can be used in the peptide probe of the present invention, provided the detectable label can be attached to the C-terminus of a peptide, directly or via a self-immolative linker, and can be detected once released from the peptide probe. Examples of other detectable labels gold nanoparticles, quantum dots, iron oxide nanoparticles, and metal complexes (e.g. copper(ll)-based, platinum(ll)-based, and ruthenium(ll)-based complexes).

[0081] In certain exemplary embodiments, the peptide probe of the present invention is NO-AH as disclosed in the Examples section herein, which has the following structure (SEQ ID NO: 3):

[0082] SEQ ID NO: 3

[0083] More specifically, in certain exemplary embodiments, the peptide probe is NO-AH as disclosed in the Examples section herein, which has the following structure (SEQ ID NO: 4):

[0084]

[0085] SEQ ID NO: 4

[0086] The peptide probe disclosed herein may be synthesised 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 portion of the peptide probes (i.e. the portion represented by Z in formula (I)) may be prepared by recombinant protein production techniques, for example, by recombinant expression of the peptide portion in bacterial, yeast, insect, fungal, plant or mammalian cells, or by cell-free in vitro peptide expression methods. The peptide portion of the peptide probe may subsequently be modified by chemical synthesis methods to install the detectable label, and when present, the N-terminal -(O)Ci-4alkyl and self-immolative linker.

[0087] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, within 1%, within 0.5%, within 0.1 %, within 0.05%, within 0.01%, within 0.005%, or within 0.001% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0088] The phrase, “consists essentially of’ and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

[0089] The peptide probes of the present invention may form salts or solvates. Salts of the peptide probes, which are suitable for use in the present invention include those wherein a counterion is pharmaceutically acceptable. However, the use of salts having non-pharmaceutically acceptable counter-ions are within the scope of the present invention, for example, when the peptide probe is only to be used in vitro, or for use as intermediates in the preparation of the peptide probe and their pharmaceutically acceptable salts and solvates, and physiologically functional derivatives thereof. Suitable salts for use according to the invention include those formed with organic or inorganic acids. In particular, suitable salts formed with acids for use according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids, or with organic sulfonic acids, such as (Ci-4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted, for example by halogen. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxalic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine. Suitable cations which may be present in salts include alkali metal cations, especially sodium, potassium and calcium, and ammonium or amino cations. In certain embodiments, the peptide probe of the present invention is in the form of a hydrochloride salt.

[0090] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. The complex may incorporate a solvent in stoichiometric or non- stoichiometric amounts. Solvates are described in Water-Insoluble Drug Formulation, 2nded R. Lui CRC Press, page 553 and Byrn et al., Pharm Res 12(7), 1995, 945-954. Before it is made up in solution, the peptide probe of the invention may be in the form of a solvate, for example, a pharmaceutically acceptable solvate. A hydrate is an example of a pharmaceutically acceptable solvate.

[0091] Compositions:

[0092] The present invention provides a composition comprising a peptide probe of the present invention and at least one binder, carrier or excipient. Preferably, the composition of the invention is a pharmaceutical composition. For example, the composition may be a pharmaceutical composition comprising a peptide probe of the present invention and at least one pharmaceutically acceptable carrier.

[0093] The composition of the invention may be suitable for oral, parenteral (including subcutaneous, intradermal, intraosseous infusion, intramuscular, intravascular (bolus or infusion), and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most suitable route may depend upon the characteristics of the subject, for example the species, age, weight, sex, medical conditions, the type and severity of the urinary tract infection that the subject has, suspected of having, or at risk of developing, and other relevant medical and physical factors. Preferably, the composition of the invention may be suitable for administration by injection and / or infusion, for example, intravascular injection or transurethral injection.

[0094] Pharmaceutically acceptable binders, carriers or excipients suitable for including in the pharmaceutical composition of the invention may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable binders, carriers or excipients may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical composition may be found in, for example, Remington The Science and Practice of Pharmacy, 19thed., Mack Printing Company, Easton, Pennsylvania (1995). Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and / or in accordance with standard and / or accepted pharmaceutical practice. It should be understood that in addition to the ingredients particularly mentioned above, the compositions for use in this invention may include other agents conventional in the art having regard to the type of composition in question. In certain embodiments, the composition comprises the peptide probe of the present invention and a pharmaceutically acceptable carrier, for example water for injection. The composition of the invention may also comprise a pharmaceutically acceptable buffer (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 of the group consisting of a polyethylene glycol (e g. PEG 400), a nonionic detergents (e g. a polysorbate, such as polysorbate- 20), and a saccharide (e.g. cyclodextrin).

[0095] Utility:

[0096] The present invention provides a method of detecting a urinary tract infection in a urine sample obtained from a subject, said method comprising: a) providing a urine sample obtained from the subject; b) contacting the urine sample with a peptide probe according to the present invention; c) determining the presence of a uropathogenic E. coli in the urine sample by detecting the detectable label of the peptide probe in the urine sample.

[0097] For the avoidance of doubt, each of steps a) to c) of the method are performed in vitro. Thus, the method of detecting a urinary tract infection in a urine sample obtained from a subject may be considered an in vitro method.

[0098] Steps a) to c) of the method of the invention may 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, step b) to c) may be performed using a urine sample obtained from a subject before the subject is administered a dose of a suitable treatment for a urinary tract infection, and then repeated using a urine sample obtained from the subject after the subject is administered a dose of suitable treatment for a urinary tract infection, for example, 1 to 7 days, 1 to 3 weeks, 1 to 3 months after the subject is administered a dose of a suitable treatment for a urinary tract infection.

[0099] As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. In embodiments, the subject is known or suspected to have a urinary tract infection, and / or is known or suspected to have a risk of developing a urinary tract infection, in particular urinary tract infection caused by uropathogenic E. coli. In certain embodiments, the subject is one that has a disease or condition known to increase the risk of developing a urinary tract infection, and / or to increase the risk of developing a more severe form of urinary tract infection. For example, the subject may be one who is known or suspected to be suffering from a disease or condition commonly associated with urinary tract infections, such as diabetes mellitus, hypertension, chronic kidney disease, dyslipidemia, ischemic heart disease, prostate disorders, and impaired immune function.

[0100] Step c) may be performed using any techniques that is suitable for detecting the detectable label present in the peptide probe of the invention once it is released from the peptide probe by the proteolytic action of OmpT and APN. For example, when the detectable label present in the peptide probe used in step c) is a fluorescence dye (e.g. a NIR fluorescence dye, such as CyOH), step c) may involve the use of fluorescence spectroscopy. In embodiments wherein the detectable label is a chemiluminescence label or a bio-luminescence label, step c) may be performed by eye (e.g. by a clinician, or any other healthcare professional) without the need of specialist equipment.

[0101] The detection and / or quantification of detectable label of the peptide probe of the invention following contact with a urine sample may accordingly be used to guide a clinician in determining a suitable disease monitoring program, suitable treatment or to inform the clinician that the subject is in remission from a urinary tract infection.

[0102] Thus, also provided herein is a method of determining one or more suitable therapeutic agents for the treatment of a urinary tract infection in a subject, said method comprising: i) providing a urine sample obtained from the subject; ii) contacting the urine sample with a peptide probe according to the present invention; iii) determining the presence of a uropathogenic E. coli in the urine sample by detecting the detectable label of the peptide probe in the urine sample; and iv) determining the one or more suitable therapeutic agents for the treatment or prevention of a urinary tract infection in the subject based on the detection of the detectable label of the peptide probe in the urine sample.

[0103] It will be evident from the above disclosure, that the peptide probe of the present invention also find utility in a method of treating or preventing a urinary tract infection in a subject. Such a method may comprise:

[0104] A. providing a urine sample obtained from the subject;

[0105] B. contacting the urine sample with a peptide probe according to the present invention;

[0106] C. determining the presence of a uropathogenic E. coli in the urine sample by detecting the detectable label of the peptide probe in the urine sample; and

[0107] D. administering to the subject a dose of one or more therapeutic agents for the treatment of the urinary tract infection based on the detection of the detectable label of the peptide probe in the urine sample.

[0108] The method of treating or preventing a urinary tract infection may comprise a step of administering a peptide probe according to the present invention to the subject, for example by transurethral injection or intravenous injection, followed by detecting the detectable label of the peptide probe in the bladder, urethra and / or urine of the subject, followed by administering to the subject a dose of one or more therapeutic agents for the treatment of the urinary tract infection.

[0109] The peptide probe of the present invention also finds utility in a method of determining one or more suitable therapeutic agents for the treatment of a urinary tract infection in a subject. Such a method may comprise administering a peptide probe according to the present invention to the subject, for example by transurethral injection or intravenous injection, and determining the one or more suitable therapeutic agents for the treatment of a urinary tract infection by detecting the detectable label of the peptide probe in the bladder, urethra and / or urine of the subject.

[0110] In the method of treating or preventing a urinary tract infection in a subject, or the method of determining one or more suitable therapeutic agents for the treatment of a urinary tract infection in a subject, the step of detecting the detectable label of the peptide probe may be performed by using any suitable detection means, for example, the detectable label may be detected in the bladder and / or urethra using in vivo imaging techniques. For example, when the detectable marker is a NIR fluorescence dye (e g. CyOH), the detectable label may be detected in the bladder and / or urethra of the subject using in vivo near-infrared fluorescence (NIRF) imaging. Additionally, or alternatively, the detectable label may be detected in urine excreted by the subject following administration of the peptide probe according into the bladder of the subject.

[0111] Suitable therapeutic agents that may be used to treat or prevent urinary tract infection include, but are not limited to, antimicrobial agents active against uropathogenic Escherichia coli. For example, suitable therapeutic agents may include an antibiotic selected from the group consisting of trimethoprim, sulfamethoxazole, fosfomycin, nitrofurantoin, p-lactam based cephalexin, ceftriaxone, cefaclor, tetracycline, and anti-microbial peptides (e.g. polymyxin B and E, and vancomycin). The dose of a suitable therapeutic agent which is required to achieve a therapeutic effect will vary with the particular route of administration and the characteristics of the subject under treatment, for example the species, age, weight, sex, medical conditions, the severity of the urinary tract infection, and other relevant medical and physical factors. An ordinarily skilled physician can readily determine and administer an effective dose of a suitable therapeutic agent required for treatment or prophylaxis of a urinary tract infection.

[0112] Kits:

[0113] The present invention also provides an in vitro diagnostic kit for use in the detection, screening, monitoring, classification, selecting treatment for, ascertaining whether treatment is working in, and / or prognostication of a urinary tract infection in a subject. The kit comprises one or more peptide probes according to the present invention, or comprising a composition of the present invention. For example, the kit may comprise the peptide probe referred to herein as NO-AH.

[0114] In certain embodiments, the kit of the present invention comprises one or more carriers (e.g. a buffer), and optionally one or more therapeutic agents suitable for treating or preventing a urinary tract infection. Examples of such therapeutic agents include those described herein.

[0115] In embodiments wherein the peptide probe present in the kit of the invention comprises a detectable label that is chemiluminescence label or a bio-luminescence label, the kit may also comprise components required to detect the label. For example, if the peptide probe present in the kit comprises luciferin as the detectable label, the kit may comprise luciferase.

[0116] In certain embodiments, the kit comprises one or more containers and may also include sampling equipment, for example, bottles, bags, vials, syringes, and test tubes. Other components may include needles, diluents, wash reagents and buffers. Typically, the kit comprises instructions, for example instructions that instruct a user to admix a stated amount of a peptide probe or composition of the present invention with a stated amount of diluent.

[0117] For the avoidance of doubt, the peptide probe or composition of the present invention, optional diluent, are present in a kit according to the present invention in a form and quantity suitable for the use of the peptide prove in a method of detection, screening, monitoring, classification, selecting treatment for, ascertaining whether treatment is working in, and / or prognostication of a urinary tract infection in a subject. The skilled person can readily determine a quantity of the peptide probe or composition of the present invention and optional diluent, suitable for the use according the present invention.

[0118] Further aspects of the present invention are defined in the following numbered clauses:

[0119] §1. A probe comprising: a) At least 5 amino acid residues; b) A self-immolative linker; and c) A dye which exhibits fluorescence only in its free form, wherein the 2 amino acid residues adjacent to the self-immolative linker are derived from dibasic amino acids (e.g. Arg and Lys).

[0120] §2. The probe according to §1 , wherein the at least 5 amino acid residues comprise the sequence (arginine, phenylalanine, phenylalanine, arginine, arginine - SEQ ID NO: 6).

[0121] §3. The probe according to §1 or §2, wherein the self-immolative linker comprises 4- aminobenzyl alcohol.

[0122] §4. The probe according to any one of §1 -§3, wherein the dye comprises a hemicyanine dye.

[0123] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The applicant reserves the right physically to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0124] Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples. EXAMPLES

[0125] As disclosed herein, the present inventors have development of a near-infrared OmpT-APN hemicyanine based fluorescence probe (referred to as NO-AH) for real-time bacterial imaging of a urinary tract infection in vitro and in vivo. As illustrated in FIG. 1 , NO-AH comprises of a hemicyanine signalling moiety that is dually locked by a dual protease activable moiety, which can be sequentially cleaved by OmpT, and then aminopeptidase N (APN). A self-immolative linker (4-aminobenzyl alcohol; PABA) between the hemicyanine reporter (CyOH) and the peptide sequence aids in the release of the free CyOH upon the consecutive cleavage of the two proteases via a 1 ,6-elimination. For UTI to occur, pathogens such as uropathogenic E. coli colonize the periurethral region of the host and urinary tract, and then ascend the urethra to enter the bladder. Uropathogenic E. coli such as CFT073 and UTI89 are known to express OmpT and APN. Thus, release of CyOH from the peptide probe, and its subsequent detection, can confirm the presence of uropathogenic E. coli in the host.

[0126] Materials:

[0127] Chemicals, solvents, and mediums

[0128] All chemicals, solvents and other reagents were used without further purification unless otherwise stated. Fmoc-protected amino acids and hexafluorophosphate benzotriazole tetramethyl uranium (HBTU) were purchased from GL Biochem (Shanghai) Ltd. N,N- Diisopropylethylamine (DIPEA), phosphorus tribromide (PBrs), 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 bought from TCI, Japan and distributed by Tee Hai Chem Pte. Ltd. Bestatin (HY-B0134-10mg) was purchased from MedChemExpress and distributed by Bio. Etc. Pte. Ltd. Luria-Bertani (LB) broth, trypticase soy broth (TSB), brain heart infusion (BHI) broth and bacto agar were bought from Becton, Dickinson and Company (Singapore) and distributed by Zuellig Pharma Pte Ltd. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin-streptomycin, trypsin-EDTA, LIVE / DEAD™ BacLight™ Bacterial Viability Kit (L7012) were purchased from Thermo Fischer Scientific.

[0129] Enzymes, bacteria strains, cell lines and mouse

[0130] E. coli BL21(DE3) was purchased from New England Biolabs Pte Ltd (Singapore). T h e wild type uropathogenic E .coli UTI89 was acquired as a gift from Professor Kimberley Kline’s lab (Singapore Centre for Environmental Life Sciences Engineering; Nanyang Technological University). E .coli CFT073 ATCC® 700928™ and E. coli ^96 (ATCC® 700336™) was bought from ATCC (USA) and supplied by Chemoscience Pte Ltd. Methicillin-resistant Staph ylococcusaureus (MRSA), Enterococcus faecalis 0G1 RF (E. faecalis) and Proteus mirabilis (P. mirabilis') were given by Prof. Qiao Yuan (Nanyang Technological University). Mouse embryonic fibroblast cell line (NIH / 3T3) were provided from American type culture collection (ATCC). Recombinant OmpT was a gift from Prof Bo Liedberg’s group. Lysozyme, Betagalactosidase (p-Gal; G4155-1 KU), nitroreductase (NTR), NADH, Caspase 3 (Casp3), Glutathione (GSH) were purchased from Sigma Aldrich. Recombinant Mouse Cathepsin B, CF (10 UG) was purchased from R&D systems and distributed by SingLab Technologies Pte Ltd. The aminopeptidase N kit (ab273292) was purchased from Abeam Singapore Pte Ltd. Female BALB / c mice (9-10 weeks old) were purchased from SLAG (Shanghai).

[0131] Instruments

[0132] Liquid chromatography mass spectrums were obtained with the Thermo Finnigan LCQ Fleet MS.1H and13C NMR spectrums were either acquired with the Bruker Advance III 400 MHz NMR, Bruker Avance II 600 MHz NMR or Bruker Avance II 700 MHz NMR. Reverse-phase high performance liquid chromatography analysis was performed with a Shimadzu HPLC system with an Alltima C-18 (250 x 10 mm) column at a flow rate of 3.0 mL / min, CH3CN (0.1 % of TFA) and water (0.1% of TFA) as the eluent. Reverse-phase high performance liquid chromatography purification was performed with Shimadzu HPLC system with an Agilent 5 prep-C18 (50 x 21.2 mm) at a flow rate of 5.0 mL / min, with CH3CN (0.1% of TFA) and water (0.1 % of TFA) as the eluent. Absorbance and fluorescence spectrums were obtained with TECAN Spark (Mannedorf, Switzerland) multimode microplate reader. Cell viability was measured by Thermo Scientific Varioskan™ LUX Multimode Microplate Reader. Confocal laser scanning microscope images were taken with the Carl Zeiss LSM 800 confocal laser microscope (Germany). Flow cytometry analysis was performed with the BD LSRFortessa X- 20 flow cytometer. Animal fluorescence imaging was performed with an I VI S Lumina II (Caliper Life Sciences) imaging system.

[0133] Chemical synthesis and characterization:

[0134] Synthesis of R-CyOH

[0135] Synthesis of R-CyOH was performed following the synthesis method set out in FIG. 2. Further details are included below:

[0136] Synthesis of CyOH: CyOH was synthesized from a reported protocol (R. Yan, Y. Hu, F. Liu, S. Wei, D. Fang, A. J. Shuhendler, H. Liu, H. Y. Chen, D. Ye, J. Am Chem Soc, 2019, 141 , 10331). Resorcinol (110.1 mg; 1.0 mmol) was first dissolved in acetonitrile (CH3CN; 3 mL) in a round bottom flask. K2CO3(138.2 mg; 1.0 mmol) was then added and stirred for 20 minutes under N2 atmosphere at 35 °C. Then, a solution of IR775-chloride in CH3CN (5 mL) was added in the round bottom flask via a syringe and stirred for 6 h at 50 °C. After that, the solution was evaporated under reduced pressure and the crude product was purified with silica gel chromatography using CH2CI2 / 0-10% CH3OH as the eluent to yield the product CyOH as a blue-green solid (250 mg; 65%).1H NMR (400 MHz, Methanol-d4) 6 8.48 (d, J = 14.1 Hz, 1H), 7.60 (s, 1 H), 7.49 (dd, J = 7.4, 2.8 Hz, 1 H), 7.38 (d, J = 6.5 Hz, 2H), 7.23 (d, J = 7.8 Hz, 2H),

[0137] 6.74 (dd, J = 8.8, 2.3 Hz, 1 H), 6.60 (t, J = 3.6 Hz, 1 H), 6.06 (d, J = 14.1 Hz, 1 H), 3.59 (s, 3H),

[0138] 2.75 (d, J = 5.9 Hz, 2H), 2.68 (d, J = 6.7 Hz, 2H), 1.90 (s, 2H), 1.75 (s, 6H).13C NMR (100 MHz, Methanol-d4) 5 174.9, 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: Calcd. For m / z, C26H26NO2 [M-CI]+384.20; Found: 384.29. HRMS (ESI) Calcd. For m / z, C26H26NO2: 384.1964 [M-CI]+; Found: 384.1966.

[0139] Synthesis of Fmoc-Arq(Pbf)-PABA (Compound 1): Fmoc-Arg(Pbf)-OH (260 mg; 0.4 mmol) was first dissolved in anhydrous CH2CI2 (5 mL), and then EEDQ (396 mg; 1.6 mmol) was added into the solution, and the mixture was stirred for 30 minutes at room temperature under N2atmosphere. After 30 minutes, PABA (197 mg; 1.6 mmol) was dissolved in anhydrous CH2CI2 (2 mL) and added in via a syringe. The mixture was stirred for 16 h at room temperature under N2atmosphere. Upon completion, the solvent was evaporated under reduced pressure. Pure compound 1 was obtained after purification with reversed-phase HPLC which yielded a white solid (110 mg; 36%).1H NMR (400 MHz, Methanol-d4) 5 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 = 6.6 Hz, 2H), 2.95 (s, 2H), 2.56 (s, 3H), 2.49 (s, 3H), 2.05 (s, 3H), 1.80 (d, J = 13.7 Hz, 1 H), 1.69 (dd, J = 9.0, 4.8 Hz, 1H), 1.42 (s, 6H).13C NMR (100 MHz, Methanol-d4) 6 157.1 , 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: Calcd. For m / z, C4I H47N5O7S [M+H]+754.32; Found: 754.46. HRMS (ESI) Calcd. For m / z, C4IH47N5O7S: 754.3274 [M+H]+; found: 754.3282.

[0140] Synthesis of Fmoc-Arg(Pbf)-PAB-CvOH (Compound 2): Pure compound 1 (52.8 mg; 0.07 mmol) was dissolved in anhydrous THF and cooled in an ice bath under N2 atmosphere. PBrs (13.3 L; 0.14 mmol) was added dropwise slowly via a syringe. The mixture was stirred for 2 h. Upon completion, the mixture was concentrated under reduced pressure. The resulting crude was dissolved in ethyl acetate (30 mL), washed with saturated sodium bicarbonate (30 mL x 3) and then brine (30 mL x 1), and dried over with anhydrous Na2SO4and concentrated under reduced pressure. The crude was used immediately in the next step of reaction without further purification. Then, CyOH (6.7 mg; 0.0175 mmol) and N,N-Diisopropylethylamine (DI PEA, 12 pL, 0.07 mmol) were first dissolved and stirred in anhydrous CH3CN at 55 °C under N2 atmosphere for 30 minutes. After that, the crude (57 mg; 0.07 mmol) was added in and the reaction mixture continued to stir at 55 °C for 6 h under N2 atmosphere. Upon completion, the resulting mixture was evaporated under pressure. Pure compound 2 (9.7 mg; 51%) was obtained after purification with reversed-phase HPLC which yielded a blue solid. 1 H NMR (400 MHz, DMSO-d6) 6 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, 1 H), 7.53 (dd, J = 10.9, 8.4 Hz, 2H), 7.40 (dd, J = 8.3, 5.5 Hz, 4H), 7.31 (h, J = 3.9 Hz, 3H), 7.24 (d, J = 8.4 Hz, 1 H), 5.38 (d, J = 5.4 Hz, 1 H), 4.27 (t, J = 6.1 Hz, 2H), 4.21 (t, J = 7.2 Hz, 2H), 4.13 (q, J = 8.3 Hz, 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, 1 H), 1.81 (t, J = 5.9 Hz, 1H), 1.74 (s, 2H), 1.38 (s, 6H). 13C NMR (100 MHz,

[0141] DMSO-d6) 6 178.3, 171.5, 171.2, 161.8, 160.6, 159.3, 158.9, 158.6, 158.2, 157.9, 156.5,

[0142] 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,

[0143] 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,

[0144] 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: Calcd. For m / z, C67H7iNsO8S [M-CI]+1119.50; Found: 1119.57. HRMS (ESI) Calcd. For m / z, [C67H7IN6O8S]+CI- 1155.4821 [M+H]+; found: 1155.4823.

[0145] Synthesis of Arq-PAB-CvOH (Compound 3; R-CvOH): Pure compound 2 (9.7 mg; 0.01 mmol) was first dissolved in stirred in trifluoroacetic acid (TFA) / CH2Cl2 (4 mL, 50% v / v) in ice bath for 2 h. The reaction was continuously monitored by analytical HPLC. Upon completion, the reaction mixture was diluted with CH2CI2 (20 mL) and washed with saturated sodium bicarbonate (30 mL x 2). The organic phase was then dried over with anhydrous Na2SO4 and concentrated under reduced pressure to yield a blue crude solid. The blue crude solid was used immediately for the next step of reaction without further purification. The crude was dissolved in DMF (1.5 mL) and piperidine (500 pL; 5% v / v in DMF) was added. The reaction mixture was stirred at room temperature for 20 minutes, and then purified by reversed phase HPLC to afford pure compound 10. (2 mg; 31%).1H NMR (600 MHz, Methanol-d4) 6 8.74 (d, J = 15.5 Hz, 1 H), 7.68 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 7.6 Hz, 1 H), 7.56 - 7.51 (m, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 3.1 Hz, 1 H), 7.44 (d, J = 4.1 Hz, 1 H), 7.35 (s, 1H), 7.07 (d, J = 2.6 Hz, 1 H), 7.02 (dd, J = 8.8, 2.4 Hz, 1 H), 6.49 (d, J = 15.3 Hz, 1 H), 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, 1 H), 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).13C NMR (100 MHz, Methanol-d4) 6 179.7, 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: Calcd. For m / z, C39H45NSO3[M-CI]+645.35; Found: 645.40. HRMS (ESI) Calcd. For m / z, [C39H45N6O3]+C|-:681.3320 [M+H]+; found: 681.3326.

[0146] Synthesis of NO-AH (Compound 7):

[0147] The fluorescent peptide probe (NO-AH, Compound 7) was prepared by coupling the NIR hemicyanine moiety CyOH to an OmpT-APN peptide substrates (see FIG. 1 and 3). Briefly, the OmpT-APN peptide was produced by solid phase peptide synthesis. Then, a self- immolative para-aminobenzoic acid (PABA) linker was used to link the NIR hemicyanine, (CyOH) to the peptide sequence. The final product 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:

[0148] Synthesis of Ac-R(Pbf)ffR(Pbf)R(Pbf)-OH (Compound 4): Peptide Ac-R(Pbf)ffR(Pbf)R(Pbf)- OH was synthesized via a standard Fmoc based solid phase peptide synthesis (SPPS) on 2- chlorotrityl chloride resin. The coupling reaction between two amino acids and acetic acid was done with HBTU as the coupling reagent and DIPEA as basic catalyst for 2-4 hours at room temperature under nitrogen atmosphere. The reaction was allowed to last for 4 hours when coupling with Arg, and 2 hours for D-Phe and acetic acid. The Fmoc protecting group was removed by 20% piperidine in dimethylformamide (DMF) for 20 minutes. The product was then cleaved off the resin using 5% TFA in dichloromethane (DCM) for 30 minutes and precipitated in cold diethyl ether to yield compound 4. Compound 4 was then used without further purification.1H NMR (400 MHz, DMSO-d6) 5 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, 1 H), 4.25 (td, J = 8.5, 5.0 Hz, 1 H), 4.17 (q, J = 6.3, 5.1 Hz, 1H), 4.16 - 4.07 (m, 1 H), 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, 1 H), 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).13C NMR (100 MHz, DMSO-d6) 6 173.7, 172.0, 171.8, 171.4, 171.0, 170.0,

[0149] 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,

[0150] 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: Calcd. For m / z, C77Hio6Ni4Oi6S3[M+H]+1579.71 ; Found: 1579.75. HRMS (ESI) Calcd. For m / z calcd, C77H106N14O16S3: 1579.7152 [M+H]+; found: 1579.7164.

[0151] Synthesis of Ac-R(Pbf)ffR(Pbf)R(Pbf)-PABA (Compound 5): Compound 4 (790 mg; 0.5 mmol) and A / -ethoxycarbonyl-2-ethoxy-1 ,2-dihydroquinoline, EEDQ (495 mg; 2.0 mmol) were

[0152] T1 dissolved in anhydrous DCM (5 ml_) and stirred for 30 minutes at room temperature under N2 atmosphere. Subsequently, 4-aminobenzyl alcohol, PABA (246 mg; 2.0 mmol) was dissolved in anhydrous DCM and added into the mixture dropwise via a syringe at room temperature under N2 atmosphere. The reaction mixture was stirred for 16 h. After reaction, the solvent was evaporated under reduced pressure and precipitated in cold diethyl ether to afford compound 5 as a white solid (800 mg; 95%). Compound 5 was used further without any purification. ESI-MS: Calcd. For m / z, C84H113N15O16S3 [M+H]+1684.77; Found: 1684.77. HRMS (ESI) Calcd. For m / z calcd, C84H113N15O16S3: 1684.7730 [M+H]+; found: 1684.7708.

[0153] 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 pL; 0.2 mmol) was then added in dropwise slowly and the reaction mixture was left to stir at 0 °C for 2 h under N2 atmosphere. Following which, the solvent was removed under reduced pressure. The resulting crude was dissolved in DCM (100 ml_), washed with saturated sodium bicarbonate (50 mL x 3) and brine (50 mL x 1). The organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure. After which, the crude product was precipitated with cold diethyl ether (20 mL x 2) to yield an off-white solid. The crude solid was then used immediately in the subsequent step without further purification. Then, 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 into the solution. The reaction mixture was stirred at room temperature for 10 minutes under N2atmosphere. The off-white crude solid (175 mg; 0.1 mmol) was then added into the reaction mixture and stirred at 50 °C for 6 h. Upon completion, the resulting mixture was evaporated under pressure. The resulting crude solids were dissolved in DCM (50 mL) and washed with deionized water (25 mL x 2) and brine (25 mL x 1). The organic layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure. Pure compound 6 (25 mg, 48%) was then obtained after purification with reversed-phase HPLC which yield a dark blue solid.1H NMR (400 MHz, Methanol-d4) 5 8.64 (dd, J = 14.7, 4.0 Hz, 1 H), 7.78 (dd, J = 33.8, 8.5 Hz, 2H), 7.63 (d, J = 7.7 Hz, 1 H), 7.49 (s, 3H), 7.47 - 7.36 (m, 3H), 7.28 (s, 1 H), 7.16 (s, 3H), 7.12 (d, J = 7.5 Hz, 1 H), 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, 1 H), 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) 6 178.2, 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: Calcd. For m / z, CiioHi37NiSOi7S3[(M-CI)+2H]2+1025.48; Found: 1025.87. HRMS (ESI) Calcd. For m / z calcd, [CiioHi37Ni6Oi7S3]+C|-: 2085.9277 [M+H]+; found: 2085.9270.

[0154] Synthesis of Ac-RffRR-PAB-CvOH (Compound 7: NO-AH): Compound 3 (25 mg; 0.01 mmol) was first dissolved in DCM (1125 pL; 22.5% v / v) and cooled in an ice bath. Triethylsilane (125 pL, 2.5% v / v) was added, followed by TFA (3750 pL, 75% v / v) into the reaction mixture. The reaction was left to stir in an ice bath for 16 h. The reaction was monitored by analytical HPLC. Upon the completion of reaction, the solvent was evaporated under reduced pressure. Pure compound 4 (10 mg; 77%) was then obtained after purification with reversed-phase HPLC which yielded a dark blue solid.1H NMR (700 MHz, Methanol-d4) 6 8.74 (dd, J = 25.0, 15.5 Hz, 1H), 7.75 - 7.64 (m, 2H), 7.54 (d, J = 5.0 Hz, 1 H), 7.51 - 7.47 (m, 1 H), 7.48 - 7.44 (m, 2H), 7.40 - 7.35 (m, 1 H), 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, 1 H), 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) 6 179.7, 173.8, 172.1, 163.6,

[0155] 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,

[0156] 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,

[0157] 21.6, 17.7, 17.6, 14.5, 9.2. ESI-MS: Calcd. For m / z, C7iH89Ni6O8 [(M-CI)+2H]2+647.35; Found: 647.63. HRMS (ESI) Calcd. For m / z calcd, [C7iH89Ni6O8]+CI-: 1329.6816 [M+H]+; found: 1329.6809.

[0158] In vitro experiments:

[0159] Preparation of stock solutions: NO-AH was dissolved in MilliQ water to obtain a 2.5 mM stock solution. Enzyme stock solutions were either used in its commercial format or dissolved in a suitable buffer as indicated by the manufacturer, or MilliQ water.

[0160] All enzymatic assays were run in a 96-well plate in a TECAN Spark (Mannedorf, Switzerland) multimode microplate reader unless otherwise stated.

[0161] Absorbance and fluorescence measurements: NO-AH(10 pM) was incubated in the presence and absence of both OmpT (18 pg / mL) and APN (1 mU), and individually, NO-AH (10 pM) was incubated in the presence of OmpT (18 pg / mL) or APN (1 mil), at 37 °C for 2 h in PBS (pH 7.4), with a working volume of 200 pL. After which, an absorbance scan was taken, and fluorescence measurements were taken with excitation of 660 nm. All experiments were done in triplicates. Selectivity studies: NO-AH (10 pM) was incubated with the different enzymes including OmpT (18 pg / mL), APN (1 mU), Lysozyme (Lz; 18 pg / mL), p-galactosidase (p-Gal; 1 unit), Nitroreductase (NTR; 18 pg / mL), Caspase 3 (Casp 3; 0.36 mU), Cathepsin B (CtsB; 18 pg / mL) and Glutathione (GSH; 18 pg / mL), with a working volume of 200 pL, at 37 °C for 2 h in PBS (pH 7.4), with the exception of CtsB and Casp3 in their respective working buffer. NADH (10 pM) was added as a cofactor for the activation of NTR. Fluorescence measurements were taken with excitation of 660 nm and emission of 720 nm. All experiments were done in triplicates. HPLC analysis: Various vials of NO-AH (10 pM) were incubated in the presence of both OmpT and APN (1 mU), sequentially added OmpT first and then APN, OmpT (18 pg / mL) or APN (1 mU) separately, and individual vials of NO-AH (10 pM), R-CyOH (10 pM) and CyOH (10 pM) at 37 °C for 2 h in PBS (pH 7.4), with a working volume of 200 pL. After incubation, 200 pL of each mixture was then injected into a Shimadzu HPLC (acetonitrile / water) to obtain the HPLC profile at absorbance of 660 nm.

[0162] Enzyme Kinetics of individual enzymes: Various concentrations of NO-AH (2.5, 5, 10, 20, 25 pM) were incubated with OmpT (3.6 pg / mL) at 37 °C for 5 mins, at a working volume of 200 pL in PBS (pH 7.4). After incubation, the mixture was injected into a Shimadzu HPLC (acetonitrile / water) for quantification analysis. Separately, various concentrations of R-CyOH (1 , 2.5, 5, 10, 20 pM) were incubated with APN (1 mU) at 37 °C for 120 mins, at a working volume of 200 pL in PBS (pH 7.4). The initial reaction rate (pM min1) was calculated against the substrate concentrations and fitted into a Michaelis-Menten curve.

[0163] The kinetic parameters were calculated using Michaelis-Menten equation: V = Vmax x [S] (Km + [S]), where V is initial velocity, and [S] is substrate concentration. The calculated parameters are as follows:

[0164] Table 1: Calculated reaction kinetics of OmpT and APN Bacterial culture: All E. coli bacterial cells, MRSA, and P. mirabilis were cultured in Luria- Bertani (LB). E. faecalis was cultured in Brain Heart Infusion broth. All bacterial strains were first streaked on agar plates from their glycerol stocks and incubated at 37 °C for 12 - 18 h. Next, a single colony of each bacteria strain was inoculated into 5 mL of broth and incubated at 37 °C overnight at 220 rpm. After which, the bacterial cells were harvested at 4000 g for 10 minutes, washed twice with PBS and resuspended into 1 mL of PBS. The suspension was diluted accordingly to an ODeoo = 1 to give a concentration of ~108CFU / mL for the subsequent experiments.

[0165] Bacterial imaging: NO-AH (10 pM) were incubated with the different bacterial strains at 37 °C for 2 h in PBS (pH 7.4) at 220 rpm, with a working volume of 100 pL. After incubation, the bacterial cells were washed with PBS twice and fixed with 15% v / v formaldehyde. 1.5 pL of the bacterial suspension was dropped on a coverslip, and then covered by another coverslip for immobilization. Confocal laser scanning microscope images were then taken with the Carl Zeiss LSM 800 confocal laser microscope at excitation of 640 nm and emission of 700 / 30 nm. For experiments that involves inhibition, the uropathogenic E. coli were inhibited with an OmpT inhibitor (3.2 mM) with the peptide sequence, Ac-RffRr, whereby the amino acid at the PT position was replaced with a D-Arg, which was reported as an inhibitor of OmpT activity (V. Hritonenko, C. Stathopoulos, Mol Membr Biol, 2007, 24, 395), for 1 h first before NO-AH was added and incubated for another 2 h, at 37 °C in PBS (pH 7.4). Separately, the uropathogenic E. coli cells were also inhibited with Bestatin (APN inhibitor; 1 mM) for 30 minutes first, before NO-AH was added and incubated for another 2h, at 37 °C in PBS (pH 7.4). All experiments were done in triplicates.

[0166] Selective recognition of co-culture of 2 different bacteria strains: CFT073 and P. mirabilis were co-incubated with NO-AH (10 pM) at varying ratios, with a working volume of 100 pL, at 37 °C for 2 h in PBS (pH 7.4) at 200 rpm. After incubation, the bacterial cells were washed with PBS twice and fixed with 15% v / v formaldehyde. 1.5 pL of the bacterial suspension was dropped on a coverslip, and then covered by another coverslip for immobilization. Confocal laser scanning microscope images were then taken with the Carl Zeiss LSM 800 confocal laser microscope at excitation of 640 nm and emission of 700 / 30 nm. All experiments were done in triplicates.

[0167] Selective localization of co-culture of 2 different bacteria strains: P. mirabilis was first stained with Hoechst (10 pM) for 30 minutes at room temperature, followed by washing with PBS once. Subsequently, Hoechst-stained P. mirabilis was co-cultured with CFT073 with NO-AH (10 pM) in the presence or absence of APN at 37 °C for 30 minutes in PBS (pH 7.4) at 200 rpm. After incubation, the bacterial cells were washed with PBS twice and fixed with 15% v / v formaldehyde. 1.5 pL of the bacterial suspension was dropped on a coverslip, and then covered by another coverslip for immobilization. Confocal laser scanning microscope images were then taken with the Carl Zeiss LSM 800 confocal laser.

[0168] Flow cytometric analysis: CFT073 and P. mirabilis were co-incubated with NO-AH (10 pM) at varying ratios, with a working volume of 100 pL, at 37 °C for 2 h in PBS (pH 7.4) at 200 rpm. After incubation, the bacterial cells were washed with PBS twice and fixed with 15% v / v formaldehyde. 10 pL of each sample was then added into 1 ml_ of PBS and then analysed on the BD Fortessa flow cytometer. Flow cytometric analysis was performed using Flowjo.

[0169] In vivo experiments:

[0170] In vivo imaging of living mice with urinary tract bacterial infection model. All the animals were cared for and treated according to the instructions and guidelines of the IACUC (NTU IACUC, protocol number A20065). A single colony E .coll CFT073 was first inoculated into 5 mL of Tryptic Soy Broth (5 mL) and grown overnight. Next, 2 mL of the overnight culture was added into another 5 mL of TSB and grown for 2 h. After that, 2 mL of bacterial culture was harvested and resuspended in 1 mL of TSB. The ODeoo was measured to be 1.263. Then, 10 pL of this culture was added into 1 mL of TSB, which is eventually used for infecting the mice. The female BALB / c mice were anesthetized accordingly, the urethral opening of the mice was disinfected and a catheter was inserted through the urethra and into the bladder. 50 pL of the bacterial culture was injected into the bladder through the catheter. The catheter was removed, and a small hemostatic forceps was used to clamp the urethral opening. The mice were infected at different time points for 6 h, 24 h and 48 h. Each infection time point was characterized by the bacterial count, H&E staining of the bladder, urethra and kidney, and interleukin-6 levels were measured using an ELISA IL-6 kit. With the established urinary tract bacterial infection model, the female BALB / c mice were injected with E. coh CFT073 and infected for 6 h first. After that, 50 pL of NO-AH (50 pM) was injected via the bladder or tail vein at different time points post-bacterial infection. The NIRF images of living mice were obtained with MS Lumina II imaging system.

[0171] Example 1 - In vitro characterization of NO-AH (Compound 7):

[0172] The stability of NO-AH in a buffer solution was investigated (see FIG. 4). The measurement revealed a relatively consistent absorbance and fluorescence intensity after 24 hours, indicating no obvious degradation of NO-AH. The optical properties of NO-AH and its reactivity towards the synergistic proteolysis of OmpT and APN were also examined. As illustrated in FIG. 5, NO-AH (10 pM) itself exhibited two characteristic absorption peaks at 604 nm and 654 nm respectively (FIG. 5b), while a simultaneous incubation of NO-AH with OmpT and APN led to a red shift in absorbance (FIG. 5b). Moreover, there was a significant fluorescence enhancement observed (~3 folds) at 720 nm when incubated with both enzymes (FIG. 5c). As controls, minimal absorbance shift was observed for NO-AH upon individual treatments by OmpT or APN (FIG. 6a). Similarly, less fluorescence enhancement was found upon NO-AH incubation with individual enzyme (Figure 6b). These optical responses clearly ascertained the need for simultaneous cleavage of OmpT and APN to give a full fluorescence enhancement of NO-AH.

[0173] Additionally, similar bacterial enzyme reactions towards NO-AH were monitored with high- performance liquid chromatography (HPLC). As shown in FIG. 5d, when NO-AH (10 pM) was first incubated with both OmpT and APN, two peaks appeared at 9.2 mins, corresponding to the R-CyOH substrate itself, and 12.8 mins, attributing to the CyOH molecule. The sequential cleavage effect by both enzymes was also validated through HPLC analysis. As shown in FIG. 5d, when NO-AH was first incubated with OmpT for 30 mins, a new peak with a retention time of 9.2 mins was observed, which was the same as the R-CyOH substrate itself, corroborating the formation of R-CyOH upon OmpT cleavage. Subsequently, added APN was added into the same mixture and incubated for another 90 minutes, and a new peak was observed with a retention time of 12.8 mins, attributed to the CyOH molecule, signifying the release of CyOH from R-CyOH after APN proteolysis. As controls, the individual enzymes with NO-AH probe were incubated separately. As shown in FIG. 7, incubation of OmpT with NO-AH gave a peak at 9.2 mins, indicating the enzyme cleavage and R-CyOH formation. However, direct incubation of APN with NO-AH could not hydrolyze probe molecule structure to CyOH and a HPLC peak with a retention time of 9.4 mins could be observed, closed to that of the NO-AH probe itself. These results clearly demonstrated the collaborative activation of dual enzymes towards NO-AH recognition. Further analysis of the enzyme kinetics of OmpT to NO-AH, and APN with R-CyOH were also monitored with HPLC, respectively. Within 5 minutes, the NO-AH molecules were facilely cleaved to form R-CyOH when incubated with OmpT. The kinetic constants were calculated with the results showing Km= 1.54 pM and Kcat = 12.33 min1(FIG.5e and FIG. 8a). Meanwhile, within 2 hours, R-CyOH substrates could be cleaved by APN to release CyOH, yielding reasonable kinetic constants of Km= 0.382 pM and Kcat = 10.24 min-1(FIG.5f and FIG. 8b). These results suggested the promising capability for each enzyme, OmpT and APN, to selectively recognize its respective substrates, NO-AH and R-CyOH, in buffer solution. Lastly, the enzymatic selectivity of NO-AH was analyzed by individually screening OmpT and APN together with several commonly used biomolecules and components (J. Weng, Y. Wang, Y. Zhang, D. Ye, J. Am. Chem. Soc. 2021, 143, 18294-18304; T. C. Do, J. Lau, C. Sun, S. Liu, K. T. Kha, S. T. Lim, Y. Y. Oon, Y. P. Kwan, J. J. Ma, Y. Mu, X. Liu, T. J. 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, 5149-5158), such as lysozyme (Lz), beta-galactosidase (P-Gal), nitroreductase (NTR), caspase 3 (Casp3), cathepsin B (CtsB) and gluthathione (GSH) etc. As shown in FIG. 5g, incubation with these common biomolecules showed negligible fluorescence enhancement. Although some non-specific signals observed after individual interactions with OmpT or APN, our NO-AH probe still exhibited promising selectivity towards the sequential activation of bacterial enzymes of OmpT and APN.

[0174] Example 2: In vitro live bacteria imaging using NO-AH (Compound 7):

[0175] 2a) Specific and Selective Fluorescence Bacterial Imaging towards Uropathogenic E. coli

[0176] The imaging feasibility the imaging feasibility of NO-AH for potential performance in various live bacterial cultures was investigated.

[0177] E. coli CFT073, E. coli UTI89 and E. coliJQQ, three types of uropathogenic E. coli strains, with high expression of OmpT, were chosen as targeted pathogens (Desloges et al., MicrobiologyOpen, 2019, 8, e915). In contrast, methicillin-resistant Staphylococcus Aureus (MRSA), Enterococcus Faecalis (E. faecalis) and Proteus Mirabilis (P. mirabilis) were selected as controls, whereby they are typically reported pathogenic bacteria species that do not contain OmpT. Upon incubation with NO-AH (10 pM), the live bacteria cultures were subjected to confocal microscopy for fluorescent imaging analysis (Wang et al., Angew. Chem. Int. Ed. Engl. 2021 , 60, 16900-16905). As shown in FIG. 9a and 9b, strong fluorescence signals were clearly observed in CFT073, UTI89 and J96 strains as compared to others that were deficient in OmpT.

[0178] As a negative control, NO-AH probe was also incubated with E. coli strains such as BL21 , a commonly used E. co / / strain lacking OmpT expression on its outer membrane. As expected, minimal fluorescence was observed in E. coli BL21 (FIG. 10a and 10b). These optical images clearly demonstrated impressive imaging capability of NO-AH, with an exceptional selectivity towards UPECs expressing OmpT.

[0179] Moreover, we further selected CFT073 and UTI89, which are typical uropathogenic E. coli strains known to cause UTI (Schaale et al., Mucosal Immunol. 2016, 9, 124-136), to validate the enzyme activity upon treatment with an OmpT inhibitor (e g. Ac-RffRr) (Hritonenko et al., Mol. Membr. Biol. 2007, 24, 395-406). As shown in FIG. 9c and 9d, the confocal imaging showed an obvious fluorescence in CFT073 and UTI89 strains upon NO-AH incubation, however, there was significant fluorescence decrease in the presence of an OmpT inhibitor, Ac-RffRr, in the bacterial imaging after treated with NO-AH probe. This further confirms the promising specificity of the NO-AH probe towards bacteria strains expressing OmpT. Likewise, we also validated the specific enzymatic activity of APN by inhibiting APN in CFT073 and UTI89. In FIG. 9c and 9e, the fluorescence signal was found to decrease significantly in the presence of APN inhibitor (e.g. Bestatin). Unequivocally, these results demonstrated the necessity of the both proteases working in synergy to specifically activate the molecular probe of NO-AH, for enhanced fluorescence imaging in uropathogenic E. coll strains. Such enzyme specific bacterial imaging can be further quantified with the fluorescence readout for the CFT073 recognition, with the results observed as low as ~104CFU / mL (FIG. 11), comparable to most of the analysis reported previously (Mendive-Tapia etal., 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). b) Synergistic Enzyme Activation of NO-AH for Localized Imaging in Bacterial Periplasm:

[0180] In our rational design of NO-AH, one unique section was the bacterial enzymes working in synergy to specifically localize activated fluorescence probe within bacterial bodies.

[0181] As shown in FIG. 9a-d, incubation of NO-AH with OmpT-positive CFT073 strain would lead to a specific bacterial imaging at -700 nm. Intriguingly, the amplified fluorescence imaging revealed a distinct silhouette formed on the surface of CFT073 (FIG. 9f), implying the possibility of well-localized fluorescence in the periplasmic region.

[0182] Furthermore, the specific and localized imaging ability of NO-AH in a mixture of live bacterial cultures was investigated. Typically, CFT073 was first co-cultured with varying ratios of Proteus Mirabilis, a controlled strain with similar morphology but no OmpT, while maintaining a total bacteria count ~108CFU / mL. As shown in FIG. 12a, upon incubation with NO-AH (10 pM) with bacterial co-cultures, an increasing trend in fluorescence signal could be clearly observed along with the bacterial co-cultures containing more proportions of CFT073. Similarly, cell cytometry (FCM) analysis was utilized to quantify the increasing fluorescence signals (Widjaja etal., Chem. Commun. (Camb.) 2017, 53, 3330-3333; Jaber etal., ACS Cent. Sci. 2020, 6, 1698-1712), and more fluorescence corresponding to increasing ratios of CFT073 in bacterial mixtures could be observed. These fluorescence responses further ascertained the imaging selectivity and regional targeting of NO-AH towards CFT073 over Proteus Mirabilis in a mixed bacterial environment.

[0183] Motivated by these promising discoveries, we then validated if localized bacterial imaging was achievable through the sequential enzymes activity and their distribution within the bacterial cell wall of UPECs to activate NO-AH. To this end, Proteus Mirabilis (PM) was first labeled with Hoechst (10 pM), followed by co-culturing with OmpT-expressed CFT073, and then incubated with NO-AH (10 pM) for imaging analysis (FIG. 12c). The confocal microscopy images in FIG. 12d showed a clear bacteria distinction between Proteus Mirabilis (PM) and CFT073, whereby the blue signal (Hoechst) represents Proteus Mirabilis, and the red signals (NO-AH) belong to CFT073, with minimal red signal staining found in Proteus Mirabilis (PM). Such responses further confirmed the selectivity and periplasmic-localization of NO-AH towards CFT073 only.

[0184] Interestingly, a red fluorescence signal surrounding the perimeter of CFT073 was observed (FIG. 12d), highlighting the significance of the spatial distribution of both enzymes in trapping the fluorescence signal via the sequential activation of OmpT and APN, so as to contribute the localized bacterial imaging in the periplasm of CFT073.

[0185] Importantly, to further evaluate the necessity of the spatial location of both enzymes and their impact on localized imaging, interference experiments were conducted to disrupt the spatial distribution of the bacterial enzymes by introducing APN into co-cultured bacterial mixture.

[0186] Upon the addition of APN (abeam; EC 3.4.11.2) into the bacterial co-cultures, there was an increase in red fluorescence staining across the overall number of bacteria. Unlike the coculture study without APN addition in FIG. 12d, it was found that more bacteria, which have been already stained with blue fluorescence, were labelled with red fluorescence (FIG. 12e). In this scenario, it is believed that OmpT on the outer membrane of CFT073 would first interact with NO-AH to produce R-CyOH. Since the added APN was present in the culture medium, these APN would have cleaved off the arginine residue from R-CyOH to afford the free CyOH leaking into the co-cultured bacterial environment. This resulted in more red fluorescence staining, especially for OmpT-negative Proteus Mirabilis (PM), in which significant red bacterial labelling can be observed.

[0187] As such, the red fluorescence signal could no longer be effectively localized in CFT073 only (FIG. 12d and 12e), and there were more fluorescence signals overlap between the red and blue channels, as quantified by the Pearson correlation coefficient value increasing from 0.485 to 0.614 (FIG. 13) along with addition of APN into the mixed bacterial environment. This phenomenon further proved that the added APN could induce the diffusion of the free CyOH in mixed bacterial environment that eventually stained into Proteus Mirabilis (PM), interfering the initial localized imaging of UPECs. These results clearly indicated the rationale that the specific and localization imaging capability of NO-AH is largely influenced by the incorporation of both OmpT and APN located in the different spaces of the UPECs bacterial cell wall.

[0188] Furthermore, the potential interference caused by the presence of abundant metabolic enzymes from the complex cellular environment in a living system was also considered. In particular, a commonly used proteolytic enzyme, trypsin, was added into live cultures of CFT073 (OmpT (+)) and P. Mirabilis (OmpT (-)) separately, followed by their individual incubation with NO-AH (10 pM). Noteworthily, the addition of trypsin into CFT073 showed a negligible fluorescence change as compared to the NO-AH incubation with CFT073 without trypsin (FIG. 14). Moreover, the fluorescence signal of NO-AH demonstrated a consistent response in the presence of both trypsin and its inhibitor (BBI), further validating the minimal influence of metabolic enzymes on UPECs staining. As expected, P. Mirabilis with trypsin addition was comparable to the faint fluorescent emission in P. Mirabilis without trypsin. These responses indicated the minimal interference of such metabolic enzymes on the specificity of NO-AH towards OmpT expressing UPECs imaging (FIG. 14).

[0189] Moreover, throughout the entire imaging studies, the live / dead staining and cellular analysis demonstrated promising biocompatibility of NO-AH, (FIG. 15 and FIG. 16), suggesting the potential of the NO-AH probe for selective labelling of OmpT-expressed CFT073 and precise imaging of UTI in vivo.

[0190] Example 3: In vivo live mice imaging using NO-AH (Compound 7):

[0191] Motivated by the excellent in vitro responses of NO-AH, the ability of NO-AH to image bacteria in vivo was explored. A urinary tract bacterial infection mouse model was established, whereby E. coli CFT073 were directly injected into the bladder of female BALB / c mice via the urethra, infecting over prolong time duration.

[0192] After infection, the number of E. coli CFT073 in the mice was measured in the urine, bladder, urethra and kidneys. As shown in FIG. 17a, the highest amount of E. coli CFT073 was observed at 6 h post infection, and gradually decreasing over time by expulsion from the body. Meanwhile, the immune response in the relevant organs were tested and the significantly elevated interleukin-6 levels were observed at 6 h post-infection, signaling an obvious inflammation response induced by bacterial infection in living mice (FIG. 17b). Furthermore, H&E staining (FIG. 18) also showed swelling and shedding of the epithelial cells at the bladder and urethra region, with a large number of bacteria spotted at 6 h post-infection in the bladder (FIG. 18; circles), suggesting the successful UTI mouse model established in vivo within a 6 h bacterial infection period for real-time imaging by using NO-AH (FIG. 19a to c). To monitor the performance and potential fluorescence changes of NO-AH probe in vivo, NO-AH (50 pM) was directly injected into the bladder of the living mice bearing the 6 h infection model, and the mice were imaged immediately under a I VIS Lumina II imaging system at various time points post-NO-AH treatment. Upon in-bladder injection of NO-AH (50 pM) in UTI infected mice, a significant fluorescence increase could be observed which could maintain over a period of 3 hours, compared to the same probe injection into healthy mice (FIG. 19d to e). Such remarkable observation indicated the feasibility of selective response of NO-AH towards a urinary tract infection. As controls, the fluorophore CyOH (50 pM) alone was also injected directly into the bladder of living mice with or without infection, and the in vivo fluorescent imaging signals were observed to remain relatively consistent for both healthy and infected mice. No apparent fluorescence difference was observed in both the healthy and UTI infected mice (FIG. 19d to e), featuring the non-selective fluorescence staining from CyOH molecule itself, whereas our dual enzyme responsive NIR NO-AH probe could specifically respond to the UTI environment and realize regional imaging of urinary tract infection in real-time.

[0193] Inspired by such promising in vivo imaging studies, we further examined the possibility of NOAH probe for in vivo UTI imaging upon its intravenous (i. v.) injection over a range of time points (FIG. 19f) . As shown in FIG. 19g and 19h, the bladder region of the mice showed an obvious fluorescence enhancement, with the fluorescence signal increasing over 3 hours. As controls, healthy mice were given the same i.v. injection of NO-AH and there was no fluorescence enhancement observed across a period of 3 hours. Furthermore, after 4 hours, with the same i.v. NO-AH injection, the mice were then sacrificed to visualize the fluorescence in organs. FIG. 19g clearly indicated signal confined in the bladder of the infected mice (see dashed circled area). These results demonstrated that NO-AH can perform real-time imaging for localized monitoring of urinary tract infection in living animals.

[0194] Notably, throughout UTI treatment in hospitals, imprecise urine tests can easily lead to the misdiagnosis of UTI that would raise the risk of recurrence and prolong the time for treatment, potentially impacting people in terms of health complications and financial burden caused by medical costs incurred. As such, we investigated the feasibility of our NIR NO-AH probe to sensitively read out the status of UTI caused by UPECs strains. As shown in FIG. 19i, after 2 days CFT073 UPECs infection in living mice, there was no bacteria detectable in the urine sample of the mice, whereas ex vivo tissue infection analysis showed that a significant amount of CFT073 (~105CFU / g) was found remaining in the bladder and urethra regions. Meanwhile, the IL-6 levels clearly indicated an immune response after 2 days of CFT073 infection (FIG. 19j). These observations implied that a bacterial infection was still occurring in the mice, suggesting the possibility of UTI misdiagnosis caused by the imprecision in routine urine test. While, in vivo imaging by administration of NO-AH into living mice with the 2 days CFT073 infection demonstrated an obvious fluorescence signal in the bladder and urethra, which was similar to that of the fluorescence observed in the bladder and urethra of the 6 h CFT073 infected mice (FIG. 19k). These findings proved that NO-AH could directly visualize the status of UTI in vivo whereby such readout may not be possible in normal urine samples (FIG. 191).

Claims

CLAIMS1. A peptide probe according to formula (I):P-Z-L-Q(I) wherein,P is absent or -C(O)Ci-6alkyl;Z represents a peptide comprising an amino acid sequence according to formula (II): [X1]„RX3[X4]mX5(II) wherein, n is an integer from 0 to 6; m is an integer from 0 to 5; each X1is independently selected from any amino acid;X3is Arg or Lys; each X4is independently selected from any amino acid; when m is 0, Xsis absent or selected from any amino acid; and when m is from 1 to 5, X5is selected from any amino acid;L is a bond or a self-immolative linker; andQ is a detectable label; or a salt or solvate thereof.

2. The peptide probe of claim 1, wherein, when present, each X1, X4, and X5are independently selected from any canonical amino acid or a D-amino acid thereof.

3. The peptide probe of claim 1 or 2, wherein: each X1is independently selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof;X3is Arg or Lys; each X4is independently selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; when m is 0, X5is absent or selected from His, Ala, Gly, He, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof; and when m is from 1 to 5, X5is selected from His, Ala, Gly, lie, Leu, Met, Trp, Phe, Vai, Lys, Arg, Pro or a D-amino acid thereof.

4. The peptide probe of any one of claims 1-3, wherein at least one of each X1, X4, and / or X5are independently selected from any basic amino acid, for example, at least one X1is His, Lys, Arg or a D-amino acid thereof, at least one X4is His, Lys, Arg or a D-amino acid thereof, and / or X5is His, Lys, Arg or a D-amino acid thereof.

5. The peptide probe of any one of the preceding claims, wherein at least one X1is D-Phe and / or at least one X4is D-Phe.

6. The peptide probe of any one of the preceding claims, wherein Z represents a peptide consisting of an amino acid sequence according to formula (Ila):X1'X2'X3'RX5'(Ha) wherein,X1' is selected from His, Lys, Arg or a D-amino acid thereof, X2' and X3' are each independently Phe or D-Phe, and X5' is Arg or Lys; and wherein, L is a bond that links X5’ to Q, or L is a self-immolative linker that links X6' to Q.

7. The peptide probe of claim 6, wherein X1' is Arg, and X2' and X3' are each D-Phe.

8. The peptide probe of any one of the preceding claims, wherein P is -C(O)Ci-4alkyl, for example P is acetyl.

9. The peptide probe of any one of the preceding claims, wherein the peptide probe is according to SEQ ID NO: 1 :SEQ ID NO: 1 wherein,L is a bond or a self-immolative linker; andQ is a detectable label; or a salt or solvate thereof.

10. The peptide probe of any one of the preceding claims, wherein Q is selected from the group consisting of a coumarin, a naphthalimide, a xanthene, a rhodamine, a cyanine, a porphyrin, a nitrobenzofurazan, a nitrobenzofurazan, and a boron-dipyrromethene.

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

12. The peptide probe of any one of claims 1-9, wherein Q is a chemiluminescence label or a bio-luminescence label.

13. The peptide probe of claim 12, wherein Q is selected from the group consisting of luminol, luciferin, a 1 ,2-dioxetane-based chemiluminescent label, and an acridinium ester.

14. The peptide probe of any one of the preceding claims, wherein L is a linking moiety according to formula (La), (Lb), (Lc), (Ld), (Le), (Lf), (Lg) or (Lh):wherein, each Rais independently selected from the group consisting of O and NH, each Rbis independently selected from the group consisting of O, NH, -OC(O)O- and -OC(O)NH-; and vvv indicates the site of attachment of the linking moiety to Z and * indicates the site of attachment of the linking moiety to Q.

15. The peptide probe of any one of the preceding claims, wherein L is a linking moiety according to:wherein, w / 1indicates the site of attachment of the linking moiety to Z and * indicates the site of attachment of the linking moiety to Q.

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

317. The peptide probe of claim 16, wherein the peptide probe is according to SEQ ID NO: 4:

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

19. A method of detecting a urinary tract infection in a urine sample obtained from a subject, said method comprising: a) providing a urine sample obtained from the subject; b) contacting the urine sample with a peptide probe according to any one of claims 1-17; c) determining the presence of a uropathogenic Escherichia coli (E. coli) in the urine sample by detecting the detectable label of the peptide probe in the urine sample.

20. A method of determining one or more suitable therapeutic agents for the treatment of a urinary tract infection in a subject, said method comprising: i) providing a urine sample obtained from the subject; ii) contacting the urine sample with a peptide probe according to any one of claims 1- 17; iii) determining the presence of a uropathogenic E. coli in the urine sample by detecting the detectable label of the peptide probe in the urine sample; and iv) determining the one or more suitable therapeutic agents for the treatment of a urinary tract infection in the subject based on the detection of the detectable label of the peptide probe.

21. A method of treating a urinary tract infection in a subject, said method comprising:A. providing a urine sample obtained from the subject;B. contacting the urine sample with a peptide probe according to any one of claims 1- 17;C. determining the presence of a uropathogenic E. coli in the urine sample by detecting the detectable label of the peptide probe in the urine sample; andD. administering to the subject a dose of one or more therapeutic agents for the treatment of the urinary tract infection based on the detection of the detectable label of the peptide probe in the urine sample.

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

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

24. The method according to any one of claims 20-23, wherein the one or more therapeutic agents is an antibiotic, for example an antibiotic selected from the group consisting of trimethoprim, sulfamethoxazole, fosfomycin, nitrofurantoin, cephalexin, ceftriaxone, cefaclor, tetracycline, and an anti-microbial peptides.

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

26. The method according to any one of claim 22-25, wherein the urinary tract infection in the subject is caused by a uropathogenic E. coli.

27. An in vitro diagnostic kit for use in the detection, screening, monitoring, classification, selecting treatment for, ascertaining whether treatment is working in, and / or prognostication of a urinary tract infection in a subject, said in vitro diagnostic kit comprising one or more peptide probes according to any one of claims 1-17.

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

Images