Compositions and methods for sensing protease activity

Abstract

Compounds for the detection of the activity of a protease include a recognition domain / substrate having an amino acid or peptide structured to interact with the protease, a reporter molecule, and a linking group forming a covalent bond between the recognition domain and reporter molecule, wherein upon interaction of the recognition domain with the protease, the covalent bond is destroyed, rendering the reporter molecule detectable by a chemical detection device. Methods of detecting enzymatic activity of a single protease may include reacting a compound as presented herein with a protease, and identifying detectable reporter molecules with a chemical detection device. Methods of detecting enzymatic activity of a plurality of proteases may include reacting a mixture of compounds, as presented herein, that are cleaved by proteases (optionally, orthogonal proteases) and release distinct reporters with proteases, and identifying detectable reporter molecules with a chemical detection device.

Description

COMPOSITIONS AND METHODS FOR SENSING PROTEASE ACTIVITYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 636,529, filed April 19, 2024, the entire contents of which are incorporated by reference herein.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under contract EB028311 awarded by the National Institutes of Health. The government has certain rights in this invention.FIELD OF THE INVENTION

[0003] The present disclosure generally relates to compositions and methods for detection, classification, prognosis, and treatment monitoring of pathologies in a patient. More specifically, the present disclosure generally relates to compositions that for use as molecular probes (also referred to as nanosensors or sensors). The molecular probes are designed to sense the activity of target enzymes, such as proteases, and subsequently degrade or decompose to generate a unique organic compound which can be detected, quantified and correlated with the activity of said target enzyme in a patient or patient sample. The unique organic compound can be a volatile organic compound (VOC).BACKGROUND

[0004] Proteases are enzymes that break down other proteins by catalyzing the hydrolysis of peptide bonds. In the human body, there are greater than 500 distinct proteases that contribute to important processes such as coagulation, digestion, host immunity, and tissue repair and remodeling. Dysregulated protease activities can also directly contribute to disease development (for example, remodeling of the extracellular matrix during tumor development or in tissue fibrosis, dysfunction of the coagulation cascade during thrombosis or other coagulopathies, T cell killing during autoimmune diseases or conditions such asinflammatory bowel disease or transplant rejection). Therefore, methods to assay for protease activity in biological samples (e.g. cell culture, tissue / liquid biopsies), animal disease models, and humans are important for studying the role of proteases in disease pathology and for potential use to diagnose and monitor disease.

[0005] Historically, assays for protease activity have relied on chromogenic or fluorogenic probes. These compounds are comprised of a signal -producing small molecule that is colored or fluorescent (i.e. a chromophore or fluorophore) that is covalently bound to a peptide substrate through a protease-cleavable amide bond. When the probe is cleaved by its target protease, the liberated chromophore / fluorophore produces signal that is measurable via a platereader. Thus, these probes produce optical functional readouts for protease activity.BRIEF SUMMARY OF THE DISCLOSURE

[0006] Various aspects of the present disclosure are directed to compositions and methods for detection, classification, prognosis, and treatment monitoring of pathologies in a patient. More specifically, the present disclosure generally relates to compositions that for use as molecular probes (also referred to as nanosensors or sensors). The molecular probes are designed to sense the activity of target enzymes, such as proteases, and subsequently degrade or decompose to generate a unique organic compound which can be detected, quantified and correlated with the activity of said target enzyme in a patient or patient sample. The unique organic compound can be a volatile organic compound (VOC).

[0007] Various non-limiting aspects of the present disclosure are as follows.

[0008] In some instances, a first aspect of the disclosure can be described as a compound for the detection of the activity of a protease, the compound comprising a recognition domain / substrate comprising an amino acid or peptide structured to interact with the protease, a reporter molecule, and a linking group forming a covalent bond between the recognition domain and reporter molecule, wherein upon interaction of the recognition domain with the protease, the covalent bond is destroyed, rendering the reporter molecule detectable by a chemical detection device.

[0009] In some instances, a second aspect of the disclosure can be described as a compound according to the first aspect, wherein the reporter molecule is a volatile organic compound,which is optionally labeled with a radioactive / non-radioactive isotope, and is volatile after destruction of the linking group.

[0010] In some instances, a third aspect of the disclosure can be described as a compound according to the first or second aspect, wherein the linking group comprises a self- immolative group.

[0011] In some instances, a fourth aspect of the disclosure can be described as a compound according to any one of the first through third aspects, wherein the recognition domain / substrate is covalently bound to a biomolecule, a polymer, or a nanoparticle scaffold.

[0012] In some instances, a fifth aspect of the disclosure can be described as a compound according to the fourth aspect, wherein the recognition domain / substrate is covalently bound to a biomolecule, wherein the biomolecule is an amino acid, a peptide, a protein, a lipid, a carbohydrate, or a nucleic acid.

[0013] In some instances, a sixth aspect of the disclosure can be described as a compound according to the fourth aspect, wherein the recognition domain / substrate is covalently bound to a polymer, wherein the polymer is linear, branched, or cyclic; the polymer is a homopolymer or copolymer; and the polymer is a polyethylene glycol (PEG)-based polymer, a dextran, a dextrin, an acrylate-based polymer, a methacrylate-based polymer, a poly(e-caprolactone), a poly( P-amino ester), poly(lactic-co-glycolic acid), a polystyrene, any functionally modified form thereof (for example, functionally modified at one or more terminus and / or internal polymer unit with an amine, a hydroxyl, a carboxyl, a carbonyl, a N-hydroxysuccinimide ester, a maleimide, dibenzocyclooctyne, and so on), or any combination thereof.

[0014] In some instances, a seventh aspect of the disclosure can be described as a compound according to the fourth aspect, wherein the recognition domain / substrate is covalently bound to a nanoparticle scaffold, wherein the nanoparticle scaffold is an nanoparticle scaffold is an inorganic nanoparticle, an iron oxide nanoparticle, a gold nanoparticle, a polymeric nanoparticle (made from, for example, any polymer listed in the sixth aspect), a dendritic nanoparticle, a micellular nanoparticle, a porous or non-porous silicon nanoparticle, a lipid-based nanoparticle (for example, liposomes, lipidnanoparticles, lipid-polymer hybrid nanoparticles (LPNs), and so on), quantum dots, or carbon nanotubes.

[0015] In some instances, an eighth aspect of the disclosure can be described as a compound according to any one of the first through seventh aspects, wherein the compound is encapsulated in a nanoparticle or a microparticle.

[0016] In some instances, a ninth aspect of the disclosure can be described as a compound according to any one of the first through eighth aspects, wherein compound comprises more than one recognition domain / substrate.

[0017] In some instances, a tenth aspect of the disclosure can be described as a compound according to any one of the first through ninth aspects, wherein the compound comprises more than one reporter molecule.

[0018] In some instances, an eleventh aspect of the disclosure can be described as a compound according to any one of the first through tenth aspects, wherein the compound comprises more than one linker.

[0019] In some instances, a twelfth aspect of the disclosure can be described as a method of detecting enzymatic activity of a single protease, the method comprising reacting a compound according to any one of the first through eleventh aspects with a protease, and identifying detectable reporter molecules with a chemical detection device.

[0020] In some instances, a thirteenth aspect of the disclosure can be described as a method of detecting enzymatic activity of a plurality of proteases, the method comprising reacting a mixture of compounds according to any one of the first through eleventh aspects, where each of the compounds are cleavable by proteases (optionally, orthogonal proteases) to release distinct reporter molecules, and identifying the distinct reporter molecules with a chemical detection device.

[0021] In some instances, a fourteenth aspect of the disclosure can be described as a method according to the twelfth or thirteenth aspect, wherein reacting the compound with the protease or plurality of proteases is performed in vivo.

[0022] In some instances, a fifteenth aspect of the disclosure can be described as a method according to any one of the twelfth through fourteenth aspects, wherein a sample is collected from a subject administered the compound to quantify the reporter molecules.

[0023] In some instances, a sixteenth aspect of the disclosure can be described as a method according to the fifteenth aspect, wherein the sample is a breath sample, a urine sample, a stool sample, a blood sample, a sweat sample, a saliva sample, a cerebrospinal fluid sample, a semen sample, a vaginal fluid, or a tears sample.

[0024] In some instances, a seventeenth aspect of the disclosure can be described as a method according to any one of the twelfth through sixteenth aspects, wherein the compound is administered to a subject by an oral route, inhalation, an intravenous route, a subcutaneous route, an intramuscular route, an intraperitoneal injection route, an ocular route, a sublingual route, a topical route, an aural route, a rectal route, or via an implanted or applied device.

[0025] In some instances, an eighteenth aspect of the disclosure can be described as a method according to the seventeenth aspect, wherein the implanted or applied device is a microneedle patch, an osmotic pump, a subcutaneous implant, or another sustained released implant.

[0026] In some instances, a nineteenth aspect of the disclosure can be described as a method according to the twelfth or thirteenth aspect, wherein reacting the compound with the protease or plurality of proteases is performed ex vivo and a reaction solution or headspace is analyzed for the reporter molecules.

[0027] In some instances, a twentieth aspect of the disclosure can be described as a method according to the nineteenth aspect, wherein reacting the compound with the protease or plurality of proteases is performed ex vivo with proteases in tissue samples or liquid biopsy samples.

[0028] In some instances, a twenty-first aspect of the disclosure can be described as a method according to the twentieth aspect, wherein the liquid biopsy sample is a blood sample, a bronchoalveolar lavage fluid, or other bodily fluid collected from a human or animal subject.

[0029] In some instances, a twenty-second aspect of the disclosure can be described as a method according to the nineteenth aspect, wherein reacting the compound with the protease or plurality of proteases is performed ex vivo with proteases in an environmental sample.

[0030] In some instances, a twenty-third aspect of the disclosure can be described as a method according to the twenty-second aspect, wherein the environmental sample is a water sample, a plant / vegetation sample, or a soil sample.

[0031] In some instances, a twenty-fourth aspect of the disclosure can be described as a method according to the twelfth or thirteenth aspect, wherein reacting the compound with the protease or plurality of proteases is performed in vitro and a reaction solution or headspace is analyzed for the reporter molecules.

[0032] In some instances, a twenty-fifth aspect of the disclosure can be described as a method according to the twenty-fourth aspect, wherein reacting the compound with the protease or plurality of proteases is performed in vitro with proteases from a mammalian tissue culture, a microbial culture, or components in a bioreactor.

[0033] In some instances, a twenty-sixth aspect of the disclosure can be described as a method according to the twenty-fifth aspect, wherein the proteases from the mammalian tissue culture, the microbial culture, or the components in a bioreactor are recombinant and / or purified proteases.

[0034] In some instances, a twenty-seventh aspect of the disclosure can be described as a method according to the twenty-fourth aspect, wherein reacting the compound with the protease or plurality of proteases is performed in vitro with proteases in an environmental sample.

[0035] In some instances, a twenty-eighth aspect of the disclosure can be described as a method according to the twenty-seventh aspect, wherein the environmental sample is a water sample, a plant sample, or a soil sample.

[0036] In some instances, a twenty-ninth aspect of the disclosure can be described as a method according to the twenty-seventh or twenty-eighth aspect, wherein the proteases in the environmental sample are recombinant proteases.

[0037] In some instances, a thirtieth aspect of the disclosure can be described as a method according to any one of the twelfth through twenty-ninth aspects, wherein the chemical detection device is a mass spectrometer, an infrared spectrometer, an ion mobility spectrometer, an electronic nose, a carbon nanotube-based detection array, a breathalyzer device, a colorimetric VOC sensor array, a microfluidic device, a human or animal nose, an engineered microbial sensor, or any combination thereof.

[0038] In some instances, a thirty-first aspect of the disclosure can be described as a method according to the thirtieth aspect, wherein the animal is a canine or a rodent.

[0039] In some instances, a thirty-second aspect of the disclosure can be described as a method according to any one of the twelfth through thirty-first aspects, wherein the identity and abundance of the reporter molecules is indicative of a disease or health status.

[0040] In some instances, a thirty-third aspect of the disclosure can be described as a method according to any one of the twelfth through thirty-second aspects, wherein the identity and abundance of the reporter molecules is used to monitor disease progression.

[0041] In some instances, a thirty-fourth aspect of the disclosure can be described as a method according to any one of the twelfth through thirty-third aspects, wherein the identity and abundance of the reporter molecules is used for disease prognosis.

[0042] In some instances, a thirty-fifth aspect of the disclosure can be described as a method according to any one of the twelfth through thirty-fourth aspects, wherein the identity and abundance of the reporter molecules is used to monitor a treatment response and, optionally, used as a clinical endpoint.

[0043] In some instances, a thirty-sixth aspect of the disclosure can be described as a method according to any one of the twelfth through thirty -fifth aspects, wherein the identity and abundance of the reporter molecules is used to monitor microbial contamination in an environmental source, a food source, a foodstuff, or a personal care product.

[0044] In some instances, a thirty-seventh aspect of the disclosure can be described as a method according to the thirty-sixth aspect, wherein the environmental source is water, soil, or vegetation.

[0045] In some instances, a thirty-eighth aspect of the disclosure can be described as a method according to the thirty-sixth aspect, wherein the food source is a crop or livestock.

[0046] In some instances, a thirty-ninth aspect of the disclosure can be described as a method according to the thirty-sixth aspect, wherein the foodstuff is an edible food product prepared at least in part from a food source.

[0047] In some instances, a fortieth aspect of the disclosure can be described as a method according to the thirty-sixth aspect, wherein the personal care product is a skin care product, a hair care product, an oral care product, a drug or pharmaceutical, or a dietary supplement.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order that the present disclosure may be readily understood, aspects of the present disclosure are illustrated by way of examples in the accompanying drawings, in which like parts are referred to with like reference numerals throughout.

[0049] FIG. 1 is a schematic illustration showing various steps for determining the presence of one or more disorders in a patient using inhalable (or otherwise administrable) molecular probes according to various aspect of the disclosure.

[0050] FIG. 2 is a schematic illustration of a nanocarrier-based molecular probe according to various aspects of the disclosure.

[0051] FIG. 3a is schematic illustration of a general structure of a molecular probe according to various aspects of the disclosure, specifically a peptide-VOC conjugate with a self-immolative (SI) linking group, and protease-triggered VOC release.

[0052] FIG. 3b is a schematic illustration of a molecular probe according to various aspects of the disclosure, specifically peptide-VOC conjugate as in FIG. 3a, designed to sense FAP activity and the workflow for assessing function. The conjugate was reacted with FAP in a VOA vial, and the reaction solution and headspace were sampled and analyzed via mass spectrometry to identify the cleavage products.

[0053] FIG. 3c is a graphical display of a MALDI-MS analysis of a reaction solution confirming FAP cleavage of the amide bond between the peptide and SI linker.

[0054] FIG. 3d is a graphical display of a PTR-MS analysis of a reaction headspace confirming volatilization of the liberated VOC reporter with the addition of FAP.

[0055] FIG. 3e is a graphical display of an FAP concentration-dependent VOC signal produced by the conjugate (n = 3).

[0056] FIG. 3f is a graphical display of a VOC signal specifically generated by FAP activity over that of other proteases that can be active in the intestine (n = 3).

[0057] FIG. 4a is schematic illustration of a general structure of a molecular probe according to various aspects of the disclosure, specifically a peptide-VOC conjugate with a self-immolative (SI) linking group, and protease-triggered VOC release.

[0058] FIG. 4b is a schematic illustration of a molecular probe according to various aspects of the disclosure, specifically peptide-VOC conjugate as in FIG. 4a, designed to senseneutrophil elastase (or “NE”) activity and the workflow for assessing function. The VOC reporter molecule was ethanethiol with a mass of 62Da. The conjugate was reacted with NE in a VOA vial, and the reaction solution and headspace were sampled and analyzed via mass spectrometry to identify the cleavage products.

[0059] FIG. 4c is a graphical display of a MALDI-MS analysis of a reaction solution confirming NE cleavage of the amide bond between the peptide and SI linker.

[0060] FIG. 4d is a graphical display of a PTR-MS analysis of a reaction headspace confirming volatilization of the liberated VOC reporter with the addition of NE.

[0061] FIG. 4e is a graphical display of an NE concentration-dependent VOC signal produced by the conjugate (n = 3).

[0062] FIG. 4f is a graphical display of a VOC signal specifically generated by NE activity over that of other proteases (n = 3).

[0063] FIG. 5a is a schematic showing the exchange of the ethanethiol reporter molecule (V62) for the methyl salicylate reporter molecule (V 152) in a modified NE molecular probe (left), which was reacted with proteases, and the signal from liberated, volatilized reporters was measured using mass spectrometry (right). Reporter signal was specifically produced by NE activity, demonstrating the interchangeability of the reporter.

[0064] FIG. 5b is a schematic showing the exchange of neutrophil elastase peptide substrate (NEpep: Nle(O-Bzl)-Met(O)2-Oic-Abu) for a cathepsin peptide substrate (CTSpep: Leu-Arg) (left), which was reacted with proteases, and the signal from liberated, volatilized reporters was measured using mass spectrometry (right). Reporter signal was specifically produced by cathepsin B (CTSB) activity, demonstrating ease of developing probes for different proteases by simple exchange of the peptide substrate.

[0065] FIGS. 6a-c illustrate the use of volatile mass barcodes for multiplexed protease sensing. FIG. 6a is a schematic illustration of four molecular probes, each having reporter molecules with a distinct molecular mass. The 4-plex was reacted with either a single protease, as illustrated in FIG. 6b, or with a combination of proteases, as illustrated in FIG. 6c, and the signal from released VOC mass barcodes was measured via mass spectrometry. At a given acquisition time, a single sample is being analyzed, and the signal across the four mass channels represent the abundance of each reporter in that sample.

[0066] FIG. 7 is a schematic illustration of probe arrays for multiplexed sensing of protease activities ex vivo in clinical bronchoalveolar lavage fluid (BALF) samples from lung transplant patients to detect bacterial infections or chronic lung allograft dysfunction (CLAD), where different disease-indicating volatile reporter signatures are produced after reacting probe arrays with clinical BALF samples.

[0067] FIG. 8 provides volcano plots indicating which probes produced significantly reduced or elevated signal in the following comparisons: BALF from Normal vs Bacterial Infections (left), Normal vs CLAD (middle), and CLAD vs Bacterial Infections (right) (n=30 for each disease group; colored dots indicate significance (p < 0.05).

[0068] FIG. 9 provides receiver operating characteristic (ROC) curves showing the accuracy of each algorithm trained to identify patients that are normal or that have CLAD or bacterial infection based on reporter signatures generated after reacting probe arrays with BALF samples (see FIG. 8) (AUC = area under the curve; SVM = support vector machine, XGB = XGBoost).DETAILED DESCRIPTION

[0069] The following description of the embodiments is merely exemplary in nature and is in no way intended to limit the subject matter of the present disclosure, their application, or uses.

[0070] It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” include plural references unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. For example, as used in this specification and the following claims, the terms “comprise” (as well as forms, derivatives, or variations thereof, such as “comprising” and “comprises”), “include” (as well as forms, derivatives, or variations thereof, such as “including” and “includes”) and “has” (as well as forms, derivatives, or variations thereof, such as “having” and “have”) are inclusive (i.e., open-ended) and do not exclude additional elements or steps. Accordingly, these terms are intended to not only cover the recited element(s) or step(s), but may also include other elements or steps not expressly recited. Furthermore, as usedherein, the use of the terms “a” or “an” when used in conjunction with an element may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Therefore, an element preceded by “a” or “an” does not, without more constraints, preclude the existence of additional identical elements.

[0071] Various aspects of the present disclosure are directed to chemical compounds that act as molecular probes to sense disease-associated protease activities in a patient. Activitybased signatures incorporating one or multiple proteases, as described herein, is a novel and effective mechanism for detection of disease or query health status. Molecular probes according to various aspects of the disclosure have various diagnostic applications including disease detection, disease classification, disease prognosis, as a means to monitor treatment response and use as clinical endpoint. Molecular probes according to various aspects of the disclosure can be administered to a patient in various ways including, but not limited to, inhalation, intratracheally, orally (for absorption in the mouth, stomach, small or large intestine) intraperitoneally, subcutaneously, intramuscularly, intravenously, transdermally, rectally, vaginally, ocularly, otically, and nasally, or via an implanted device or an applied device (for example, a microneedle patch, an osmotic pump, a subcutaneous implant, or any other form of sustained release implant).

[0072] In some instances, molecular probes according to various aspects of the disclosure are particularly useful for the detection of lung diseases or disorders in a patient, such as, but not limited to, lung cancer, lung infections, interstitial lung diseases (ILDs) such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disorder (COPD), emphysema, alpha- 1 antitrypsin deficiency, and asthma.

[0073] In some instances, molecular probes according to various aspects of the disclosure are particularly useful for the detection of extrapulmonary disorders (i.e., disorders affecting other organs besides the lungs) in a patient, such as, but not limited to, cancers, infections, thrombosis and coagulopathies, autoimmune diseases, inflammation, injury, fibrosis, and wound healing.

[0074] In some instances, molecular probes according to various aspects of the disclosure are particularly useful for the detection of microbial contaminants in a patient. Molecular probes according to various aspects of the disclosure are also particularly useful for the detection of microbial contaminants in environmental sources such as, for example, water,soil, or vegetation, surfaces or items (for example hospital / surgical room surfaces, previously sterilized medical containers, and so on). Molecular probes according to various aspects of the disclosure are also particularly useful for the detection of microbial contaminants in food sources such as, for example, crop and livestock. Molecular probes according to various aspects of the disclosure are also particularly useful for the detection of microbial contaminants in foodstuffs. As used herein, a “foodstuff’ is an edible food product or ingestible dietary supplement made or prepared at least in part from a food source. Molecular probes according to various aspects of the disclosure are also particularly useful for the detection of microbial contaminants in personal care products such as, for example, hair care products, skin care products, oral care products, drugs or pharmaceuticals, and dietary supplements.

[0075] Proteases catalyze proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. Proteases do this by cleaving the peptide bonds within proteins via hydrolysis. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism and cell signaling. In the human body, there are greater than 500 distinct proteases that contribute to important processes such as coagulation, digestion, host immunity, and tissue repair and remodeling. Dysregulated protease activities can also directly contribute to disease development (for example, remodeling of the extracellular matrix during tumor development). Therefore, methods to assay for protease activity in biological samples (for example, cell cultures, tissue / liquid biopsies), animal disease models, and humans are important for studying the role of proteases in disease pathology and for potential use to diagnose and monitor disease.

[0076] Historically, assays for protease activity have relied on chromogenic or fluorogenic probes. These compounds are comprised of a signal -producing small molecule that is colored or fluorescent (i.e. a chromophore or fluorophore) that is covalently bound to a peptide substrate through a protease-cleavable amide bond. When the probe is cleaved by its target protease, the liberated chromophore / fluorophore produces signal that is measurable via a platereader. Thus, these probes produce optical functional readouts for protease activity.

[0077] Various aspects of the disclosure are directed to molecular probes that traffic through the body and release organic compounds (referred to below as reporter molecules) upon degradation by specific enzymes (e.g., proteases), where the organic compounds are solubilized and detectable in liquid samples. Various aspects of the disclosure are also directed to ingestible molecular probes that traffic through the body and release organic compounds upon degradation by specific enzymes, where the organic compounds are volatile (VOCs) and detectable in a vapor or gaseous form. When the organic compounds are volatile (or “volatile reporter molecules”), they may be eliminated from the body in breath and can be quantified via various gas-phase chemical detection devices and methodologies such as, for example, gas-chromatography mass spectrometry. This strategy enables disease detection via breath testing.

[0078] Breath tests are non-invasive, rapid, and can be completed with ease at great frequency and in a variety of settings for monitoring applications. In some instances, molecular probes according to various aspects of the disclosure are particularly useful in diagnostic platforms, as they are designed to degrade and as resulting degradation products can be exhaled and act as biomarker signatures indicative of a disease exhibiting a particular enzymatic activity.

[0079] Molecular probes according to various aspects of the disclosure can include three components for the detection of protease activity. A first component is a recognition domain or recognition molecule (or recognition substrate such as an amino acid or peptide) that has a chemical structure configured to bind to an active site of a specific protease. Exemplary recognition domains (substrates), their corresponding protease for activity testing, and the disease associated with a particular protease activity and provided in Tables 1 and 2.Table 1.Table 2.

[0080] A second component is a reporter molecule that is covalently bound to the recognition domain. A third component is a linking group bonding the recognition domain and the reporter molecule. When the molecular probe is intact (i.e., the recognition domain is bound to the reporter molecule via the linking group), the reporter molecule is non- detectable. When the linking group of the molecular probe is destroyed, however, the reporter molecule is converted to a detectable reporter molecule in liquid or, if volatile, in gas form. The now detectable reporter molecule exhibits a various properties such as a characteristic molecular mass. When the detectable reporter molecule is non-volatile it can be detected in various liquid-phase analytical detection devices such as liquid chromatography-mass spectrometry (LC-MS). When the detectable reporter molecule is volatile it can be detected in various gas-phase analytical detection devices such as gas chromatography-mass spectrometry (GC-MS), proton transfer reaction-mass spectrometry (PTR-MS), selected-ion flow-tube mass spectrometry (SIFT-MS), and ion mobility spectroscopy. Like naturally-occurring volatile compounds produced in the body, volatile reporter molecules will be exhaled after diffusing into blood circulation, followed by pulmonary gas exchange. Concentrations of individual volatile reporter molecules in breath can be quantified (via, for example, mass spectrometry) and used to build a classifier of disease using machine learning. Altogether, catalytic processing of volatile-releasing probes by target proteases will produce an amplified, mass-encoded disease signature fornon-invasive detection in breath. Molecular probes according to various aspects of the present disclosure, regardless of the degree of volatility of the resulting detectable reporter molecule, have utility in in both in vitro and in vivo diagnostic applications. Generally, various chemical detection devices maybe used for either gas-phase or solution-phase detection of reporter molecules in including gas chromatography-mass spectrometers, liquid chromatography-mass spectrometers, infrared spectrometers, ion mobility spectrometers, electronic noses, carbon nanotube-based detection arrays, breathalyzer devices, colorimetric VOC sensor arrays, microfluidic devices, human or animal (for example, canine and rodent) noses, engineered microbial sensors, or any combination thereof.

[0081] In some instances, the linking group is made partially or completely of a single atom. In some instances the single atom is an oxygen, nitrogen or sulfur atom. Upon destruction of the single atom linking group, the single atom forms part of the reporter molecule such as a hydroxyl (-OH) group, an amine (-NH2) group or a thiol (-SH) group. In some instances, the linking group is or comprises a protease-cleavable bond. In some instances, the protease-cleavable bond is a covalent bond.

[0082] In some instances, the linking group may include a self-immolative group. Representative molecular probe structures with an exemplary self-immolative group are provided in Formulae (I) and (II) as follows:Self-immolative linker Alcohol volatile(I)Self-immolative linker Thlolate volatile (II)

[0083] When the covalent bond connecting the self-immolative group to the recognition domain and / or the reporter molecule is cleaved, the self-immolative group self-destructs, which results in the release of the reporter. The liberated reporter molecule recovers its characteristic mass and optional volatility. Exemplary Formulae (I) and (II), both contain a peptide recognition domain. Formula (I) contains a reporter molecule with an oxygen atom bound to the self-immolative group, which results in the formation of a reporter molecule comprising a hydroxyl group after destruction of the self-immolative group. Formula (II) contains a reporter molecule with a sulfur atom bound to the self-immolative group, which results in the formation of a volatile reporter molecule comprising thiol group after destruction of the self-immolative group. Another exemplary formula contains a reporter molecule with a nitrogen atom bound to the self-immolative group, which results in the formation of a reporter molecule comprising an amine group after destruction of the self-immolative group.

[0084] In some instances, the recognition domain / substrate of molecular probes according to the disclosure can further be covalently bound to a biomolecule such as, for example an amino acid, a peptide, a protein, a lipid, a carbohydrate, or a nucleic acid.

[0085] In some instances, the recognition domain / substrate of molecular probes according to the disclosure can further be covalently bound to a nanoparticle scaffold, wherein the nanoparticle scaffold is an nanoparticle scaffold is an inorganic nanoparticle, an iron oxide nanoparticle, a gold nanoparticle, a porous or non-porous polymeric nanoparticle (made from, for example, any polymer listed in the sixth aspect), a dendritic nanoparticle, a micellular nanoparticle, a porous or non-porous silicon nanoparticle, a lipid-based nanoparticle (for example, liposomes, lipid nanoparticles, lipid-polymer hybrid nanoparticles (LPNs), and so on), or a carbon based nanoparticle (for example, single- or multiwalled carbon nanotubes, carbon dots, graphite, graphene, activated carbon, and so on).

[0086] In some instances, the recognition domain / substrate of molecular probes according to the disclosure can further be covalently bound to a polymer. The polymer can be linear, branched, or cyclic. The polymer can be a homopolymer or copolymer. The polymer can be made of various polymers including, but not limited to, polyethylene glycol (PEG)- based polymers, dextrans, dextrins, acrylate-based polymers, methacrylate-basedpolymers, poly(s-caprolactones), a poly(P-amino esters), poly(lactic-co-glycolic acids), polystyrenes, any functionally modified form thereof (for example, functionally modified at one or more terminus and / or internal polymer unit with an amine, a hydroxyl, a carboxyl, a carbonyl, a N-hydroxysuccinimide ester, a maleimide, dibenzocyclooctyne, and so on), or any combination thereof.

[0087] In some instances, sensing the activity of multiple proteases can be accomplished using a plurality of ingestible molecular probes according to various aspects of the disclosure. A biomarker signature comprised of the activity of multiple proteases, for example, provides superior disease specificity over that of a single protease. For multiplexed sensing of protease activities, probe arrays are needed in which each probe senses a distinct protease and is barcoded with a distinct VOC reporter. In the original probes, VOCs were directly conjugated to peptide substrates via an amide bond. Reliance on this original chemistry limits the degree of multiplexing (i.e. the number of proteases that can be sensed at one time). Therefore, new strategies are presented herein to covalently attach diverse VOCs to peptide substrates. In particular, many VOCs with the generally- regarded as safe (GRAS) FDA designation contain hydroxyl and thiol functional groups and have well-characterized toxicity profiles. Thus, incorporation of GRAS VOCs is ideal for probes that will be used to assay for in vivo protease activities.

[0088] FIG. l is a schematic illustration showing various exemplary steps for determining the presence of one or more disorders in a patient using ingestible molecular probes according to various aspect of the disclosure. In FIG. 1, the recognition domain (or “substrate”) can be, for example, an amino acid / peptide when the molecular probe is for use to detect protease activity.

[0089] In some instances, molecular probes according to various aspects of the disclosure can be administered via inhalation or intratracheal instillation to sense protease activities in the lungs. In some instances, molecular probes according to various aspects of the disclosure can be administered in via alternative administration routes (e.g. intravascular, intraperitoneal, subcutaneous injections) to deliver molecular probes to other tissue or organs of the patient. When the molecular probes distribute to tissues and are cleaved, the resulting reporter molecules, will diffuse into blood circulation and can be measured via blood sample analysis. Alternatively or additionally, when the molecular probes arereleased in tissues and cleaved, the resulting reporter molecules, will diffuse into blood circulation and may be exhaled after pulmonary gas exchange. In other instances, the resulting reporter molecules may present and measurable in other bodily fluids such as, for example, urine, stool, sweat, saliva, cerebrospinal fluid, semen, vaginal fluid, or tears.

[0090] In some instances, molecular probes according to various aspects of the disclosure can be mixed with biological and / or clinical samples (for example, pure proteases, bacterial / mammalian cell cultures, cell / tissue homogenates and supernatants, biopsies, blood, stool, bronchoalveolar lavage fluids, or other bodily fluids) to assay for enzymatic activity. Probe cleavage can be monitored by measuring the concentration of reporters molecules in a solution or gas using, for example, LC-MS or GC-MS, respectively. As discussed elsewhere herein, other chemical detection devices may also be used. Potential applications include microbial pathogen identification, in-line monitoring for cell manufacturing processes, and for tissue engineering.

[0091] In some instances, molecular probes according to various aspects of the disclosure can be used for evaluation of a disease or health status of a subject. In some instances, molecular probes according to various aspects of the disclosure can be used to monitor disease progression in a subject. In some instances, molecular probes according to various aspects of the disclosure can be used for disease prognosis. In some instances, molecular probes according to various aspects of the disclosure can be used to monitor a treatment response and, optionally, used as a clinical endpoint.

[0092] In some instances, molecular probes according to various aspects of the disclosure can be mixed with environmental samples (for example, water, sewage, soil, plants / vegetation) to assay for enzymatic activity. In some instances, molecular probes according to various aspects of the disclosure can be mixed with food source (for example, crops and livestock), foodstuff (for example, edible food products prepared at least in part from a food source), or personal care product (for example skin care products, hair care products, oral care products, drugs, pharmaceuticals, dietary supplements, and so on) samples to assay for enzymatic activity. In some instances, molecular probes according to various aspects of the disclosure can be mixed with bioreactor samples to assay for enzymatic activity. Probe cleavage can be monitored by measuring the concentration of reporters molecules in a solution or gas using, for example, LC-MS or GC-MS,respectively. As discussed elsewhere herein, other chemical detection devices may also be used. Potential applications include microbial pathogen identification in such samples.

[0093] Alternative molecular probes according to various aspects of the disclosure are also envisioned herein. For example, in some instances, molecular probes may comprise a singular or plurality of reporter molecules bound to a biocompatible polymeric nanoparticle (or “nanocarrier”) via an amino acid or peptide linker(s) (FIG. 2). When reporter molecules are bound to the nanocarrier, they cannot be detected. Upon cleavage of the linker by target proteases, the reporter molecules are released and recover their characteristic mass, and optional volatility, for detection in liquid or vapor form.

[0094] Molecular probe engineering as an alternative to biomarker discovery. Diverse biological species (e.g. proteins, nucleic acids, metabolites, circulating tumor cells) have been identified as biomarkers of disease. However, reliance on these naturally-occurring biomarkers for disease identification is limiting due to fundamental biological and technical challenges such as low concentrations, noise from biological background, instability, and difficulties in their isolation from complex biological matrices (e.g. blood, stool). Moreover, biomarker type dictates the tools and methods used for biomarker quantification. Thus, there are limited degrees of freedom for optimizing biomarker diagnostic performance. Therefore, rather than rely on biomarker discovery to identify naturally-occurring biomarkers of enzymatic disorders, The present invention allows for logical engineering of ingestible molecular probes de novo by leveraging known disease biology and newly-engineered enzyme-responsive molecules. Using this approach, we are no longer at the mercy of what Nature provides, but have greater control over factors contributing to biomarker signal -to-noise ratio such as (1) choice of measurable reporter molecule(s), (2) kinetics of biomarker(s) production via molecular probe design, and (3) probe dosing regimen(s). Furthermore, we can optimize for specificity by engineering multiplexed biomarkers. This approach allows one to fully leverage the benefits of breath testing in clinical diagnostics, as breath biomarker discovery has had limited success to date.

[0095] Plug-and-play chemistry to induce the exhalation of diverse VOCs in breath. A handful of prior art clinical breath tests for diseases rely on metabolic processing of volatile organic compound (VOC) precursors (small molecules or macromolecules) intovolatile biomarkers for exhalation. These include the 13C-urea breath test for H. pylori infection, the 13C-methacetin breath test for liver function, and the small intestinal bacterial overgrowth (SIBO) breath test in which compounds are ingested and metabolized by disease activity into VOCs. These breath tests induce the exhalation of a limited range of VOCs in breath - 13C isotope-labeled carbon dioxide, hydrogen, and methane. In contrast to the VOC precursors used in existing breath tests, molecular probes according to various aspects of the disclosure are highly modular, and volatile reporter molecules formed therefrom are exchangeable. In accordance with various aspects of the disclosure, hydroxyl-, thiol- and amine-containing VOCs can be used as volatile reporter molecules and can be covalently bound to a recognition domain via a cleavable bond. We have already identified hundreds of VOCs with generally regarded as safe (GRAS) FDA designation containing such functional groups that can be incorporated into molecular probes according to various aspects of the disclosure. GRAS compounds are highly benign molecules, many of which are already used as food flavorings and should have minimal regulatory barriers to translation. Using plug-and-play chemistry to generate a library of specific recognition domains (e.g., specific activity proteases) covalently bound to correspondingly distinct reporter molecules (which are converted to volatile reporter molecules exhibiting, for example, distinguishable molecular masses) as described above, various and diverse molecular probes can be generated to query specific and multiple types of protease activity.

[0096] Ultrasensitive detection via protease-catalyzed signal amplification. Breath biomarker signal is amplified via protease-driven reporter release. In this process, protease activity is not consumed, and a single protease can cleave thousands of molecular probes per hour, thus, driving signal amplification for sensitive tumor detection. In the context of volatile reporter molecules, sensitivity is further enhanced through breath sampling methods that concentrate the resulting volatile reporter molecules in large volumes of breath collected over time, which can be collected onto, for example, sorbent tubes for downstream analysis via mass spectrometry.

[0097] A significant feature of the present invention is the ability to synthesize different molecular probes, where each is designed to sense a different target protease and release distinct reporter molecules exhibiting a distinct mass. This strategy should produce breath biomarker signatures that offer greater disease specificity than existing single biomarkers.Protease targets will be nominated based on literature search and analysis of transcriptional datasets. Exemplary protease targets are provided in Tables 1 and 2.

[0098] Due to the interchangeability of the peptide substrate in the probes, protease specificity is easily tuned. To identify peptides cleaved during a specific disease state, a library of fluorogenic peptide substrates can be screened against recombinant and / or purified proteases in 384-well plate format. Fluorescence from peptide cleavage can be measured over time using a plate reader. Hierarchical cluster analysis of cleavage rates can be used to eliminate redundant substrates and downselect for a peptide panel that is cleaved by proteases (optionally, orthogonal proteases). The peptides can then be barcoded with volatile reporters of distinct mass. Volatile reporters can be covalently bound to peptides via self-immolative linkers, as described elsewhere herein. Self-immolative linkers can adjoin the C-terminus of peptide recognition domains to diverse functional groups frequently found in VOCs (e.g. hydroxyl, thiol, and amine groups). In this format, we have shown that peptide cleavage triggers the linker to self-immolate (i.e. self-destruct), thereby converting reporter molecules to corresponding volatile reporter molecules without any linker remnants that could reduce their volatility. Barcoded peptides can be purified via HPLC, and the product mass can be confirmed with LC-MS. Protease-triggered volatile reporter molecule release can be confirmed by reacting barcoded peptides with their respective proteases and quantifying volatilized reporters in the reaction headspace using a proton transfer reaction-mass spectrometer (PTR-MS). VOC-barcoded peptide substrates can suffice alone as probes or can be conjugated onto nanocarriers or encapsulated for in vivo delivery. To minimize barriers to in-human testing, probe components will be restricted to compounds safe for administration in humans. This includes the use of organic compounds with Generally Regarded As Safe (GRAS) FDA designation as reporter molecules. To date, we have identified more than 200 GRAS VOCs that are compatible for use in accordance with various aspects of the disclosure. Other components include peptides and polyethylene glycol nanocarriers, which are components in diagnostic nanoparticles that have undergone successful phase I clinical trials for safety.

[0099] An important consideration for in vivo molecular probe function is in vivo stability. In some instances, encapsulation of the molecular probe(s) may be necessary due to potential instability of the probe in certain tissues (for example, in the acidic and proteolyticenvironment of the stomach). Similar to drugs that require protection from the acidic and proteolytic gastric environment, probes can be encapsulated in pH-responsive microparticle formulations using microspraying methods for oral delivery. pH-responsive formulations will enable controlled release of nanosensors in the small intestine (which is more alkaline than the stomach in both humans and mice). In some instances according to this example, microparticle formulations will incorporate Eudragit®, a copolymer of methacrylic acid and ethyl acrylate, which is insoluble at gastric pH but soluble at intestinal pH and therefore useful to protect drugs that are unstable in gastric fluid or to prevent off- target effects in the gastric mucosa. Spray-drying methods have been previously used to synthesize pH-responsive microparticles incorporating Eudragit®. Such methods will be investigated and pharmacokinetic and biodistribution studies completed to determine the transit time and controlled release of probe formulations through the small and large intestine. Prior studies have shown that a number of breath tests have conserved function in humans, mice and other rodent models due to overlapping disease biology and VOC elimination pathways. Therefore, we plan to validate our approach in mice.

[0100] Example 1 - VOC conjugation to peptides for protease sensing

[0101] VOCs (volatile reporter molecules) that are covalently bound to the cleavage site of peptide substrates are released upon peptide cleavage. Liberated VOCs undergo phase transition into a gas to produce a signal quantifiable by mass spectrometry. In this example, linking group chemistries, specifically the use of self-immolative linkers, are established to incorporate diverse VOCs into peptide-VOC conjugates for multiplexing. Self- immolative linkers can adjoin the C-terminus of peptide substrates to functional groups frequently found in VOCs (e.g., hydroxyl and thiol groups), in addition to amine groups. In this format, peptide cleavage will trigger the linker to self-immolate (i.e. self-destruct), thereby releasing the VOC reporter without any linker remnants that could reduce its volatility (FIGS. 3a and 4a).

[0102] The feasibility of this approach has been confirmed by synthesizing a conjugate (molecular probe) containing a peptide substrate for fibroblast activation protein a (FAP) and methyl salicylate (a safe-to-ingest, hydroxyl-containing VOC with a mass-to-charge (m / z) ratio of 153) (FIG. 3b). FAP is an ECM-remodeling protease with elevated expression in cancer-associated fibroblasts and helps with tumor cell invasion in CRC andother cancers. Elevated FAP expression has been associated with worse clinical outcomes, and the protease has garnered widespread attention as a promising pan-cancer biomarker and therapeutic target. Thus, FAP is a highly relevant protease target for CRC detection. Characterization of VOC reporter release from peptide-VOC conjugates is possible via in vitro cleavage assays, where conjugates are reacted with proteases in volatile organic analysis (VOA) vials, and gastight syringes are used to pierce the rubber septa caps for headspace sampling (FIG. 3b). For the FAP-sensing conjugate, reaction products confirmed cleavage of the amide bond between the FAP peptide substrate and the self- immolative linker (FIG. 3c) and vaporization of the released methyl salicylate reporter (FIG. 3d). In vitro cleavage studies further confirmed FAP concentration-dependent reporter signal (FIG. 3e) and specific sensing of FAP activity over that of other proteases (FIG. 3f). In this example, we will build upon this preliminary work and systematically test conjugation of different VOC reporters to our model peptide substrate for FAP.

[0103] The feasibility of this approach has also been confirmed by synthesizing a conjugate (molecular probe) containing a peptide substrate for neutrophil elastase (NEpep: Nle(O-Bzl)-Met(O)2-Oic-Abu, or “NE”) and ethanethiol (a safe-to-ingest, thiol-containing VOC with a mass of 62 Da) (FIG. 4b). When the probe was reacted with purified NE, analysis of solubilized and vaporized cleavage products via mass spectrometry confirmed that the bond between the peptide and self-immolative linker was cleaved and that the linker underwent self-destruction to release the volatilized reporter (FIGS. 4c-d). In subsequent cleavage assays with NE, we showed that the magnitude of signal from liberated, volatilized reporters reflects the level of neutrophil elastase activity (FIG. 4e). Protease specificity is important when assaying for the activity of a particular protease in more complex biological samples that contain multiple proteases. In additional cleavage assays, we showed that production of reporter signal is only triggered by NE activity and not by other protease activities (FIG. 4f).

[0104] By holding the peptide substrate constant and exchanging the reporter molecule, the modularity of this approach will be tested for VOC reporters with different functional groups and overall structure (aliphatic versus aromatic). Since VOCs are distanced from the cleavage site by the self-immolative linker, we expect that VOC chemical structure will have a lesser effect on Michaelis-Menten cleavage kinetics than if they were directlyconjugated to the peptide. The reaction rate constant (kcat) and Kmwill be determined for each conjugate for comparison of catalytic efficiency. Exemplary VOC reporter molecules with the Generally Regarded as Safe (GRAS) FDA designation have been identified for testing (Table 3). GRAS compounds are commonly used as food flavorings and are, therefore, safe for ingestion and have well-characterized toxicity profiles. For future translation in humans, we expect patients to fast 12 hours before testing to minimize background signal from food volatiles, which is common practice for clinical breath tests.Table 3.

[0105] Example 2 - Modular probe chemistry enables incorporation of diverse peptide substrates and volatile reporters

[0106] Having shown that new probe format with self-immolative linkers retains the ability for protease sensing, we next sought to demonstrate the interchangeability of reporters and substrates. A second neutrophil elastase probe was synthesized with a different VOC reporter - methyl salicylate (Fig. 5a). This probe retained its ability to sense neutrophil elastase activity with high protease specificity and demonstrates the ease of incorporating VOCs with hydroxyl functional groups in addition to previously-shown thiol-containing VOCs (Fig. 4b). Of note, all reporters used in the working examples are GRAS compounds and are safe for in vivo use. The amino acid sequence of peptide substrates dictates which proteases will cleave the probe. Therefore, we sought to determine if the peptide component in the new probe format could be easily exchanged to develop probes for diverse proteases. We synthesized a third probe by replacing the peptide substrate for neutrophil elastase with a substrate for cathepsins (Fig. 5b). With the modification, the probe could no longer sense neutrophil elastase activity, but could sense the activity of cathepsin B, a type of cathepsin. Altogether, these results demonstrate the ease of synthesizing probes that can be used to assay diverse protease activities with easily-tailored volatile reporters.

[0107] Example 3 - Multiplexing via VOC mass barcodes

[0108] Past studies have shown that 14 and 19-plex nanosensor panels provide sufficiently broad coverage of cancer-associated protease activity for early detection of lung and prostate cancers in mouse models with 100% sensitivity and 95% specificity. Therefore, using self-immolative linker chemistry, a 15-20-plex nanosensor panel will be created for detection of each disease of interest. In preliminary multiplexing studies, a 4-plex was synthesized by coupling 4 different peptide substrates for Proteases A, B, C, and D to 4 VOC reporters of distinct mass (FIG. 6a). In in vitro cleavage assays, the 4-plex was either reacted with a single protease (FIG. 6b) or a combination of proteases (FIG. 6c). These studies show that the activity of multiple proteases could be monitored simultaneously via measurement of mass-encoded reporter signals in the reaction headspace. These preliminary studies confirm multiplexed sensing in vitro, which is a crucial milestone towards engineering breath biomarker signatures.

[0109] To achieve a larger panel of 15-20 nanosensors, 15-20 VOC reporters with optimal physicochemical properties for trafficking from the GI tract to the breath will be identified. Through review of publications from the Flavor Extract Manufacturers Association (FEMA), more than 200 GRAS compounds with suitable functional groups for coupling to self-immolative linkers have been identified and rank-ordered on their vapor pressure, logP (i.e., hydrophobicity), and airwater partition coefficients. The top 20 VOC reporter candidates are shown in Table 3. These will be assessed empirically via dosing of compound mixtures in mice via oral gavage and subsequent measurement of exhaled reporters at multiple timepoints from 5 min to Ih after dosing. VOCs traffic from their tissue of origin into breath in minutes in both humans and mice. In both species, VOCs diffuse from their tissue of origin into systemic circulation and are exhaled following pulmonary gas exchange. Due to conserved VOC elimination pathways, breath tests have comparable function in humans and mouse models of disease. Therefore, mice are a suitable models to assess our approach. Breath samples will be collected and analyzed using methods we have previously established in which mice are placed in sealed chambers for a fixed time and the chamber headspace is subsequently displaced into evacuated glass vials for analysis via mass spectrometry. For increased detection sensitivity, breath volatiles will be collected in sorbent tubes. Using these methods, mouse breath can readily be analyzed to identify optimal VOC mass barcodes for multiplexing.

[0110] Example 4 - Multiplexed sensing of protease activities ex vivo in clinical bronchoalveolar lavage fluid (BALE) samples

[0111] In this example, molecular probes according to various aspects of the disclosure were used as a probe array for multiplexed sensing of protease activities ex vivo in clinical bronchoalveolar lavage fluid (BALF) samples from lung transplant patients to detect bacterial infections or chronic lung allograft dysfunction (CLAD). FIG. 7 is a schematic illustration of probe array for multiplexed sensing of protease activities ex vivo in clinical bronchoalveolar lavage fluid (BALF) samples from lung transplant patients to detect bacterial infections or chronic lung allograft dysfunction (CLAD), where different diseaseindicating volatile reporter signatures produced after reacting probe arrays with clinical BALF samples, and Table 4 provides compositional details of each molecular probe used in the probe array in this Example.Table 4.

[0112] FIG. 8 provides volcano plots indicating which probes produced significantly reduced or elevated signal in the following comparisons: BALF from Normal vs Bacterial Infections (left), Normal vs CLAD (middle), and CLAD vs Bacterial Infections (right) (n=30 for each disease group; colored dots indicate significance (p < 0.05). Each dot in the volcano plot represents one probe at one measurement timepoint (vPX-Y, where X = 1-8 for probe number (see Table 4) and Y indicates the time at which volatile reporters were measured - after 2 or 4 h reaction of the probe array with the BALF sample). FIG. 9 provides receiver operating characteristic curves showing the accuracy of each algorithm trained to identify patients that are normal or have CLAD or bacterial infection based on reporter signatures generated after reacting probe arrays with BALF samples (see FIG. 8) (AUC = area under the curve; SVM = support vector machine, XGB = XGBoost).

[0113] While certain implementations have been described in terms of what may be considered to be specific aspects, the present disclosure is not limited to the disclosed aspects. Additional modifications and improvements to the aforementioned vial adapter may be apparent to those skilled in the art. Moreover, the many features and advantages of the disclosure are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the present disclosure which fall within the spirit and scope of the disclosure.

Claims

CLAIMSWhat is claimed is:

1. A compound for the detection of the activity of a protease, the compound comprising: a recognition domain / substrate comprising an amino acid or peptide structured to interact with the protease; a reporter molecule; and a linking group forming a covalent bond between the recognition domain and reporter molecule, wherein upon interaction of the recognition domain with the protease, the covalent bond is destroyed, rendering the reporter molecule detectable by a chemical detection device.

2. The compound of claim 1, wherein the reporter molecule is a volatile organic compound, which is optionally labeled with a radioactive / non-radioactive isotope, and is volatile after destruction of the linking group.

3. The compound of claim 1, wherein the linking group comprises a self-immolative group.

4. The compound of claim 1, wherein the recognition domain / substrate is covalently bound to a biomolecule, a polymer, or a nanoparticle scaffold.

5. The compound of claim 4, wherein the recognition domain / substrate is covalently bound to a biomolecule, wherein the biomolecule is an amino acid, a peptide, a protein, a lipid, a carbohydrate, or a nucleic acid.

6. The compound of claim 4, wherein the recognition domain / substrate is covalently bound to a polymer, wherein the polymer is linear, branched, or cyclic, the polymer is a homopolymer or copolymer,the polymer is a polyethylene glycol (PEG)-based polymer, a dextran, a dextrin, an acrylate-based polymer, a methacrylate-based polymer, a poly(s-caprolactone), a poly( P- amino ester), poly(lactic-co-glycolic acid), a polystyrene, any functionally modified form thereof, or any combination thereof.

7. The compound of claim 4, wherein the recognition domain / substrate is covalently bound to a nanoparticle scaffold, wherein the nanoparticle scaffold is an iron oxide nanoparticle, a gold nanoparticle, a polymeric nanoparticle, a dendritic nanoparticle, a micellular nanoparticle, a porous or non-porous silicon nanoparticle, a lipid-based nanoparticle, a quantum dot, or a carbon nanotube.

8. The compound of claim 1, wherein the compound is encapsulated in a nanoparticle or a microparticle.

9. The compound of claim 1, wherein the compound comprises more than one recognition domain / sub strate .

10. The compound of claim 1, wherein the compound comprises more than one reporter molecule.

11. The compound of claim 1, wherein the compound comprises more than one linker.

12. A method of detecting enzymatic activity of a single protease, the method comprising: reacting a compound according to any one of claims 1 to 11 with a protease; and identifying detectable reporter molecules with a chemical detection device.

13. A method of detecting enzymatic activity of a plurality of proteases, the method comprising: reacting a mixture of compounds according to any one of claims 1 to 11, where each of the compounds are cleavable by proteases to release distinct reporter molecules; andidentifying the distinct reporter molecules with a chemical detection device.

14. The method of claims 12 or 13, wherein reacting the compound with the protease or plurality of proteases is performed in vivo.

15. The method of any one of claims 12 to 14, wherein a sample is collected from a subject administered the compound to quantify the reporter molecules.

16. The method of claim 15, wherein the sample is a breath sample, a urine sample, a stool sample, a blood sample, a sweat sample, a saliva sample, a cerebrospinal fluid sample, a semen or vaginal fluid sample, or a tears sample.

17. The method of any one of claims 12 to 16, wherein the compound is administered to a subject by an oral route, inhalation, an intravenous route, a subcutaneous route, an intramuscular route, an intraperitoneal injection route, an ocular route, a sublingual route, a topical route, an aural route, a rectal route, or via an implanted device or an applied device.

18. The method of claims 12 or 13, wherein reacting the compound with the protease or plurality of proteases is performed ex vivo and a reaction solution or headspace is analyzed for the reporter molecules.

19. The method of claim 18, wherein reacting the compound with the protease or plurality of proteases is performed ex vivo with proteases in tissue samples or liquid biopsy samples.

20. The method of claim 19, wherein the liquid biopsy sample is a blood sample, a urine sample, a bronchoalveolar lavage fluid, or other bodily fluid collected from a human or animal subject.

21. The method of claim 18, wherein reacting the compound with the protease or plurality of proteases is performed ex vivo with proteases in an environmental sample.

22. The method of claim 21, wherein the environmental sample is a water sample, a plant sample, or a soil sample.

23. The method of claims 12 or 13, wherein reacting the compound with the protease or plurality of proteases is performed in vitro and a reaction solution or headspace is analyzed for the reporter molecules.

24. The method of claim 23, wherein reacting the compound with the protease or plurality of proteases is performed in vitro with proteases from a mammalian tissue culture, a microbial culture, or components in a bioreactor.

25. The method of claim 24, wherein the proteases from the mammalian tissue culture, the microbial culture, or the components in a bioreactor are recombinant or purified proteases.

26. The method of claim 23, wherein reacting the compound with the protease or plurality of proteases is performed in vitro with proteases in an environmental sample.

27. The method of claim 26, wherein the environmental sample is a water sample, a plant sample, or a soil sample.

28. The method of claim 26 or 27, wherein the proteases in the environmental sample are recombinant proteases.

29. The method of any one of claims 12 to 28, wherein the chemical detection device is a mass spectrometer, an infrared spectrometer, an ion mobility spectrometer, an electronic nose, a carbon nanotube-based detection array, a breathalyzer device, a colorimetric VOC sensor array, a microfluidic device, a human or animal nose, an engineered microbial sensor, or any combination thereof.

30. The method of claim 29, wherein the animal is a canine or a rodent.

31. The method of any one of claims 12 to 30, wherein the identity and abundance of the reporter molecules is indicative of a disease or health status.

32. The method of any one of claims 12 to 30, wherein the identity and abundance of the reporter molecules is used to monitor disease progression.

33. The method of any one of claims 12 to 30, wherein the identity and abundance of the reporter molecules is used for disease prognosis.

34. The method of any one of claims 12 to 30, wherein the identity and abundance of the reporter molecules is used to monitor a treatment response and, optionally, used as a clinical endpoint.

35. The method of any one of claims 12 to 30, wherein the identity and abundance of the reporter molecules is used to monitor microbial contamination in an environmental source, a food source, a foodstuff, or a personal hygiene product.

36. The method of claim 35, wherein the environmental source is water, soil, or vegetation37. The method of claim 35, wherein the food source is a crop or livestock.

38. The method of claim 35, where the foodstuff is an edible food product prepared at least in part from a food source.

39. The method of claim 35, wherein the personal care product is a skin care product, a hair care product, an oral care product, a drug or pharmaceutical, or a dietary supplement.

40. The method of claim 17, wherein the implanted or applied device is a microneedle patch, an osmotic pump, a subcutaneous implant, or other sustained release implant.

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