WO2020215043A1 - Advanced glycation end-product breaking biocatalysts - Google Patents

Abstract

In various aspects and embodiments the invention provides compositions and methods for removing glucosepane from one or more proteins in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, or a biologically active fragment thereof.

Description

TITLE OF THE INVENTION

Advanced Glycation End-Product Breaking Biocatalysts CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application Serial No.

62/836,382, filed April 19, 2019, the content of which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 5R21AI124116-02 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

The present application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created April 18, 2020, is named 047162-7176WO1_ST25.txt and is 55.1 kilobytes in size. BACKGROUND OF THE INVENTION

Advanced Glycation End-Products (AGEs) are protein post-translational

modifications associated with aging, diabetes, and several degenerative diseases.

The most common AGE in human tissues is a protein crosslink called glucosepane, which is a lysine-arginine protein cross-linking product derived from D-glucose.

Glucosepane is present in human tissues at levels 10 to 1000 times higher than any other cross-linking AGE, and is currently considered to be the most important cross-linking AGE.

Chemically, glucosepane is known as (2S)-2-Amino-6-((6R,7S)-2-(((S)-4-amino-4- carboxybutyl)amino)-6,7-dihydroxy-6,7,8,8a-tetrahydroimidazo[4,5-b]azepin-4(5H)- yl)hexanoic acid. As an AGE, the reaction pathway that leads to glucosepane formation is known as the Maillard Reaction, or non-enzymatic browning.

Glucosepane is an irreversible, covalent cross-link product that makes intermolecular and intramolecular cross-links with a wide variety of proteins, including collagen of the extracellular matrix (ECM) and crystallin of the eyes. Covalent protein cross-links irreversibly link proteins together in the ECM of tissues. Levels of glucosepane cross-links in human collagen in the ECM increases progressively with age and at a more rapid pace in people with diabetes, thus suggesting the role of glucosepane in the long-term effects associated with diabetes and aging such as arteriosclerosis, joint stiffening, and skin wrinkling. Further, the build-up of cross-links such as glucosepane within and between proteins is shown to reduce proteolytic degradation in the ECM. This leads to increased cross-link accumulation and is thought to be linked to the thickening of basement membranes in capillaries, glomeruli, lens, and lungs.

There is thus a need in the art for methods and compositions capable of reversing glucosepane crosslinks. This disclosure addresses that need. BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of removing glucosepane from a subject’s tissue. In certain embodiments, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a biologically active fragment thereof.

In another aspect, the invention provides a method of treating or preventing an advanced glycation end-product associated (AGE-associated) disease in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a biologically active fragment thereof.

In another aspect, the invention provides a method of treating or preventing an advanced glycation end-product associated (AGE-associated) disease in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13, or a biologically active fragment thereof.

In another aspect, the invention provides a method of removing N^e- (carboxyethyl)lysine (CEL) or N^e-(carboxymethyl)lysine (CML) from a subject’s tissue. In certain embodiments, the method comprises administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13, or a biologically active fragment thereof.

In another aspect, the invention provides a pharmaceutical composition comprising a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, or a biologically active fragment thereof, wherein the composition is formulated for topical or ocular administration.

In certain embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-11.

In certain embodiments, the tissue comprises the extracellular matrix of a tissue. In certain embodiments, the tissue comprise a basement membrane of the extracellular matrix of a tissue. In certain embodiments, the tissue comprises a basement membrane (such as, but not limited to, blood vessel (such as, but not limited to, a capillary), glomeruli, lens, and/or lung). In certain embodiments, the tissue comprises joint tissue. In certain embodiments, the tissue comprises skin.

In certain embodiments, the polypeptide further comprises at least one selected from the group consisting of a cell-penetrating peptide, a cell secretion signaling peptide, and a stability enhancing domain.

In certain embodiments, the polypeptide is formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

In certain embodiments, the composition is formulated for topical or ocular administration.

In certain embodiments, the polypeptide is administered topically or ocularly to the subject.

In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of illustrative embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG.1A depicts that an arginine auxotrophic strain of E. coli (DArgA) overexpressing HemF is capable of breaking down and using glucosepane as an arginine source while the same strain overexpressing GFP as a negative control is not.

FIG.1B depicts that HemF degrades glucosepane in vitro.

FIG.1C depicts phenylisothiocyanate (PITC) derivatization of in vitro HemF assays and showing that the enzyme releases citrulline through its cleavage of glucosepane.

FIG.2A depicts a phylogenetic tree showing human HemF and human HemF microbiome homologs.

FIG.2B depicts relative degradation of glucosepane by eight HemF homologs compared to incubation with no enzyme.

FIG.3 depicts that glucosepane is degraded by varying amounts when incubated with the discovered enzymes. Glucosepane is not degraded when no enzyme is present or when incubated with the purported "crosslink-breaking" compound ALT-711.

FIG.4A depicts growth of E. coli lysine auxotroph in M9 minimal medium supplemented with lysine, CML, CEL, or water vehicle control.

FIG.4B depicts the identification of CEL-cleaving biocatalyst through transposon mutagenesis.

FIG.5A-5B depict an analogy between the transformation of cmnm⁵(s²)U to nm⁵(s²)U by C-terminal domain of MnmC (C-MnmC) and the restoration of lysine from CEL. FIG.5A depicts two consecutive reactions catalyzed by the bifunctional tRNA-modifying enzyme MnmC in E. coli. C-MnmC catalyzes FAD-dependent demodification of carboxymethyl group in cmnm⁵(s²)U that generates primary amine which is methylated by SAM-dependent N-MnmC. FIG.5B depicts lysine restoration from CEL cleavage. In both transformations, demodification of carboxymethyl- or carboxyethyl- functionality generates a primary amine.

FIGs.6A-6D depict the validation of C-MnmC-mediated CEL cleavage using a-keto acid derivatization assay. FIG.6A depicts the mechanism of deamination by D-amino acid oxidases (DAAO). FIG.6B depicts a proposed mechanism of CEL cleavage and the formation of 2,4-DNPH-pyruvate hydrazone adduct. MnmC exhibits hydrogen peroxide scavenging activity (FIG.11). FIG.6C depicts the detection of CEL cleavage-dependent hydrazone adduct formation spectrophotometrically. The adduct appears red-brown in color and its absorbance spectrum shows l_m at 445 nm, and a shoulder at 515 nm. FIG.6D depicts liquid chromatography-mass spectrometry (LC-MS) validation of MnmC-dependent generation of syn- and anti-isomers (*) of the hydrazone adduct (m/z 267, negative ion mode).

FIG.7A depicts relative activities of MnmC variants. Standalone WT C-MnmC shows about 5-fold improvement in CEL-cleaving activity.

FIG.7B depicts the steady-state kinetics of WT MnmC and WT C-MnmC showing concentration dependencies with CEL.

FIG.7C depicts the detection of CML cleavage-dependent 2,4-DNPH-glyoxylic acid hydrazone adduct formation spectrophotometrically. The adduct appears orange in color and its absorbance spectrum shows l_max at 455 nm.

FIG.7D depicts the quantitation of the amount of glyoxylic acid generated from CML cleavage by C-MnmC.

FIGs.8A-8C depict a comparison of the crystal structures of MnmC (PDB: 3PS9, orange) and D-amino acid oxidase from human (PDB: 2DU8, grey). FIG.8A depicts a superposition of MnmC and D-amino acid oxidase. FIG.8B depicts the active site of the C- terminal domain of MnmC (indicated by red arrow). FIG.8C depicts the active site of D- amino acid oxidase (indicated by red arrow). FAD is shown in green.

FIGs.9A-9C depict the activity of WT C-MnmC on a peptidomimetic substrate DKP- CEL and a peptide substrate Pept-CEL. FIG.9A depicts the structures of DKP-CEL and Pept-CEL. FIG.9B depicts the activity of C-MnmC on DKP-CEL relative to its activity on free CEL. FIG.9C depicts LC-MS detection of C-MnmC-mediated cleavage of Pept-CEL and the release of DEF-K-ADE (*).

FIGs.10A-10B depict a representative tandem mass spectra for pre- and post- cleavage of CEL functionality by C-MnmC. FIG.10A depicts HRMS/MS spectrum for pre- cleavage substrate DEF-(CEL)-ADE showing fragments that contain CEL. FIG.10B depicts an HRMS/MS spectrum for post-cleavage product DEF-K-ADE showing fragments that contain lysine instead of CEL. Addition of carboxyethyl functionality on a lysine side chain corresponds to an increase of 72 units in mass, and this difference is observed between fragments detected in pre- and post-cleavage tandem MS spectra.

FIGs.11A-11B depict hydrogen peroxide scavenging activity by MnmC. FIG.11A depicts hydrogen peroxide (40 mM) incubated in presence and absence of MnmC at room temperature and 37 ^oC. No significant change in the hydrogen peroxide concentration is observed without MnmC. In presence of MnmC, dose-dependent reduction in hydrogen peroxide concentration is observed. FIG.11B depicts hydrogen peroxide (40 mM) incubated in presence of C-MnmC or BSA control. Significant reduction in the hydrogen peroxide concentration is observed only in presence of C-MnmC.

FIG.12 depicts a cmnm⁵U₃₄ binding model of MnmC.10 residues around the bound substrate were chosen for mutational analysis. Mutated residues are shown in green.

cmnm⁵U₃₄ is shown in blue. FAD is shown in yellow.

FIG.13A-13B depict a calibration curve for 2,4-DNPH-pyruvate hydrazone adduct. FIG.13A depicts UV-Vis absorption spectra for 2,4-DNPH-pyruvate hydrazone adduct were obtained using increasing concentrations of pyruvate. FIB 13B depicts a calibration curve used to determine the amount of pyruvic acid generated from CEL cleavage.

FIG.14 depicts the proposed mechanism of CML cleavage and the formation of 2,4- DNPH-glyoxylic acid hydrazone adduct. C-MnmC-mediated cleavage of CML generates glyoxylic acid which is derivatized by 2,4-DNPH and basified to generate a hydrazone adduct which has a distinctive orange color (in contrast to red-brown color for pyruvate adduct) and absorbance maximum at 455 nm.

FIG.15A-15B depict the calibration curve for 2,4-DNPH-glyoxylic acid hydrazone adduct. FIG.15A illustrates UV-Vis absorption spectra for 2,4-DNPH-glyoxylic acid hydrazone adduct were obtained using increasing concentrations of glyoxylic acid. FIG.15B illustrates calibration curve used to determine the amount of glyoxylic acid generated from CML cleavage.

FIG.16 depicts a comparison of the active sites in C-MnmC (PDB: 3PS9) (left) and RebH (PDB: 2OA1) (right). In both active sites, there is a chloride ion (green) is near the alloxazine ring of FAD.

FIG.17 depicts homologs of C-MnmC and their active sites (indicated by red arrow). Amadoriases I & II from Aspergillus fumigatus and D-amino acid oxidase from Rhodotorula gracilis exhibit large and exposed active sites whereas glycine oxidase from Bacillus subtilis and D-amino acid oxidase from pig kidney exhibit small and buried active sites. DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplified materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

As used herein, the term "composition" or "pharmaceutical composition" refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, ocular, intra-ocular, aerosol, parenteral, ophthalmic, pulmonary and topical administration.

An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.

As used herein, a "basement membrane" is a thin, fibrous, extracellular matrix that separates the lining of an internal or external body surface from underlying connective tissue in animals. This surface may be epithelium (skin, respiratory tract, gastrointestinal tract, and so forth), mesothelium (pleural cavity, peritoneal cavity, pericardial cavity, and so forth), and/or endothelium (blood vessels, lymph vessels, and so forth). The basement membrane and the interstitial matrix comprise the extracellular matrix (ECM).

The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human. As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term "pharmaceutically acceptable carrier" means a

pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;

glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid;

pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference. As used herein, the term "preventing" a disease or disorder means preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition.

As used herein, "topical administration" or "topical application" refers to a medication applied to body surfaces such as the skin or mucous membranes.

As used herein, "treating a disease or disorder" means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.

As used herein, the term "treatment of " or "treating" a state, disorder or condition includes: inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or relieving the disease, i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Compounds & Compositions

In one aspect, the invention provides a polypeptide selected from the group consisting of SEQ ID NOs: 1-13, as well as a composition comprising an effective amount of a polypeptide selected from the group consisting of SEQ ID NOs: 1-13.

Without wishing to be limited by theory, in certain embodiments, the polypeptides of the invention catalyze glucosepane cleavage in vitro (FIG.1B). In various embodiments, the polypeptide is 7-carboxy-7-deazaguanine synthase-like (QueE-like) enzyme, a

coproporphyrinogen III oxidase-like (HemF-like) enzyme, a 5,6-dimethylbenzimidazole synthase-like (BluB-like) enzyme, a methylenetetrahydromethanopterin reductase-like enzyme, or a putative radical SAM domain-containing enzyme, or any biologically active fragment thereof. In some embodiments, the polypeptide is a homolog of HemF. In various embodiments, the polypeptide is selected from the group consisting of SEQ ID NOs: 5-11.

In various embodiments, the polypeptide further comprises a heterologous peptide. In various embodiments, the heterologous peptide comprises a cell penetrating peptide, by way of non-limiting example a transactivator of transcription (TAT) peptide, a secretion signal peptide, by way of non-limiting example a preprotrypsin signal sequence, or a stability enhancing peptide. In various embodiments, the stability enhancing peptide can be any peptide that extends the polypeptide’s half-life in vivo relative to the polypeptide alone.

In various embodiments, the polypeptide is formulated as a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient. In various embodiments, the composition is formulated for topical and/or ocular administration.

In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:1. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:2. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:3. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:4. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:5. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:6. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:7. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:8. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:9. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:10. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:11. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:12. In various embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO:13.

In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:1. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:2. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:3. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:4. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:5. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:6. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:7. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:8. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:9. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:10. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:11. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:12. In various embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:13. 7-carboxy-7-deazaguanine synthase (QueE)

EC number: 4.3.99.3

Nucleotide sequence: SEQ ID NO: 14

Amino acid sequence - SEQ ID NO: 1

Putative radical SAM enzyme

(Unknown function, no EC number)

Nucleotide sequence: SEQ ID NO: 15

Amino acid sequence - SEQ ID NO: 2

5,6-dimethylbenzimidazole synthase (BluB) EC number: 1.14.99.40

Nucleotide sequence: SEQ ID NO: 16

Amino acid sequence - SEQ ID NO: 3

Coproporphyrinogen III oxidase (CPOX/HemF)

EC number: 1.3.3.3

Nucleotide sequence: SEQ ID NO: 17

Amino acid sequence - SEQ ID NO: 4

Coproporphyrinogen III oxidase (HemF) Homologs (EC number 1.3.3.3 for all below) Acetobacteraceae

Nucleotide sequence: SEQ ID NO: 18

Amino acid sequence - SEQ ID NO: 5

Brevundimonas

Nucleotide sequence: SEQ ID NO: 19

Amino acid sequence - SEQ ID NO: 6

Afipia

Nucleotide sequence: SEQ ID NO: 20

Amino acid sequence - SEQ ID NO: 7

Human (N-terminus truncated) Nucleotide sequence: SEQ ID NO: 21

Amino acid sequence - SEQ ID NO: 8

Ralstonia

Nucleotide sequence: SEQ ID NO: 22

Amino acid sequence - SEQ ID NO: 9

Escherichia

Nucleotide sequence: SEQ ID NO: 23

Amino acid sequence - SEQ ID NO: 10

Pseudomonas

Nucleotide sequence: SEQ ID NO: 24

Amino acid sequence - SEQ ID NO: 11

In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of a polypeptide selected from the group consisting of SEQ ID NOs: 12 and 13 or a catalytically active fragment thereof. Without wishing to be limited by theory, in certain embodiments, the polypeptides of the invention catalyze the cleavage of CEL and CML (FIGs.4A and 4B; FIGs.6A-6D). In various embodiments, the polypeptide is Mnmc or the C-terminal domain on Mnmc (C-Mnmc). As described in Example 2, below, the C- terminal domain of Mnmc catalyzes the degradation of CEL and CML, both when fused to Mnmc N-terminal domain and as an isolated fragment (C-Mnmc). As shown below, C- Mnmc is more catalytically active with respect to AGE’s than full length Mnmc, however a skilled person will recognize that both are within the scope of the invention. Methods of Cleaving Glucosepane from a Peptide and Treating or Preventing Disease In one aspect, the invention provides a method of removing glucosepane from one or more proteins in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or any biologically active fragments thereof.

In another aspect, the invention provides a method of treating or preventing an advanced glycation end-product (AGE) associated disease, the method comprising administering to the subject an effective amount of a composition comprising a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 1- 13, or any biologically active fragments thereof.

Glucosepane is a stable post-translational protein modification associated with aging and age-related disease, and the compositions of the invention can be applied to reduce levels of glucosepane in a subject in need thereof. In various embodiments the AGE-associated disease can be Alzheimer’s disease, diabetes, atherosclerosis, joint stiffening, skin wrinkling, and//or thickening of basement membranes in capillaries, glomeruli, lens (such as, but not limited to, in cataracts and/or presbyopia), and/or lungs. In various embodiments the subject is a mammal. In various embodiments the subject is a human.

In another aspect, the invention provides a method of treating or preventing an AGE- associated disease, the method comprising administering to the subject an effective amount of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, or any biologically active fragments thereof. In various embodiments, the polypeptides of the invention are employed to remove AGE’s and treat and/or prevent AGE- associated disease. In various embodiments, the AGE is at least one selected from the group consisting of glucosepane, CEL, and CML. In various embodiments, the AGE-associated disease can be Alzheimer’s disease, diabetes, atherosclerosis, joint stiffening, skin wrinkling, and//or thickening of basement membranes in capillaries, glomeruli, lens (such as, but not limited to, in cataracts and/or presbyopia), and/or lungs.

In another aspect, the invention provides a method of removing CEL or CML from one or more proteins in the tissue of a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, or any biologically active fragments thereof. MnmC

Nucleotide sequence: SEQ ID NO: 25

Amino acid sequence: SEQ ID NO: 12

C-MnmC

Nucleotide sequence: SEQ ID NO: 26

Amino acid sequence: SEQ ID NO: 13

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat or prevent a disease or disorder in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat or prevent a disease or disorder in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the

determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of a disease or disorder in a patient.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

In certain embodiments, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

Compounds of the invention for administration may be in the range of from about 1 µg to about 10,000 mg, about 20 µg to about 9,500 mg, about 40 µg to about 9,000 mg, about 75 µg to about 8,500 mg, about 150 µg to about 7,500 mg, about 200 µg to about 7,000 mg, about 350 µg to about 6,000 mg, about 500 µg to about 5,000 mg, about 750 µg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packaged

pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second

pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents.

Routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Ocular/Topical Administration

An obstacle for topical administration of pharmaceuticals is the stratum corneum layer of the epidermis. The stratum corneum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limit the penetration rate (flux) of a compound through the stratum corneum is the amount of the active substance that can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Such formulations may be applied to the skin directly or through the use of swabs, applicators, spatulas and the like, as well as in the form of transdermal patches. In certain embodiments, the patch minimizes loss of pharmaceuticals through washing, friction, scratching and/or rubbing of the skin. In other embodiments, the patch increases absorption of the pharmaceutical through the skin, while minimizing the exposure of the skin to the pharmaceutical.

Topically administrable formulations contemplated within the invention may, for example, comprise from about 0.001% to about 1% (w/w) active compound, although the concentration of the active compound may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram,

alkanecarboxylic acids, polar lipids, dimethylsulfoxide, or N-methyl-2-pyrrolidone.

One acceptable vehicle for topical delivery of some of the compositions of the invention may contain liposomes. The composition of the liposomes and their use are known in the art (for example, U.S. Patent No.6,323,219).

In alternative embodiments, the topical formulation further comprises other ingredients such as adjuvants, anti-oxidants, chelating agents, surfactants, foaming agents, wetting agents, emulsifying agents, viscosifiers, buffering agents, preservatives, and the like. In other embodiments, a permeation or penetration enhancer is included in the formulation and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum corneum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2- pyrrolidone, are known to those of skill in the art. In another aspect, the topical formulation may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum corneum, and thus allows increased transport across the stratum corneum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art.

Additional non-active ingredients in the topical formulation are well known in the art. These ingredients include, but are not limited to, humectants, emollients, pH stabilizing agents, chelating agents, gelling agents, thickening agents, emulsifiers, binders, buffers, carriers, anti-oxidants, etc. Additional examples of such ingredients are included in the U.S. Food & Drug Administration, Inactive Ingredients for Approved Drugs, available online. Addition discussion and potential non-active ingredients that may be included in formulations can be found in "The Science and Practice of Pharmacy", 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa. (2006).

Parenteral Administration

For parenteral administration, the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos.6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790.

Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466;

20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In one embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on

Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

In the case wherein the patient’s status does improve, upon the doctor’s discretion the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection. Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD₅₀ and ED₅₀. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application. EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Example 1:

Advanced Glycation End-Products (AGEs) are protein posttranslational modifications associated with aging, diabetes, and several degenerative diseases. A strategy to reverse the formation of AGEs and restore the native structure to proteins therefore has significant therapeutic use. To this end, a functional metagenomics approach was undertaken to find enzymes from soil bacteria that can break the most common AGE protein crosslink, which is known as glucosepane. Five enzymes that allow a DArgA auxotroph E. coli strain to utilize free synthetic glucosepane as an arginine source (FIG.1A) have been uncovered. These enzymes include a 7-carboxy-7-deazaguanine synthase-like (QueE-like) enzyme, a coproporphyrinogen III oxidase-like (HemF-like) enzyme, a 5,6-dimethylbenzimidazole synthase-like (BluB-like) enzyme, a methylenetetrahydromethanopterin reductase-like enzyme, and a putative radical SAM domain-containing enzyme.

The ability of four of these enzymes (heterologously expressed and isolated HemF, BluB, QueE, and the putative radical SAM domain-containing enzyme) to degrade synthetic glucosepane over time in vitro (FIG.1B) has been validated. It has also been demonstrated through amino acid derivatization of enzymatic reactions that HemF-catalyzed cleavage of glucosepane releases citrulline as one of the products (FIG.1C), which E. coli then processed to arginine, explaining the observed restoration of growth in an auxotroph screening strain overexpressing HemF.

HemF homologs are widely distributed in the environment, not only among bacteria but in animals (including humans) as well (FIG.2A). For this reason, the ability of HemF homologs to degrade glucosepane was explored and several were found that could do so with similar activity to the environmental enzyme (FIG.2B). These enzymes include the human HemF homolog, as well as homologous bacterial proteins from genera found in relevant human microbiome sites: Escherichia (gut microbiome), Pseudomonas (gut microbiome), Brevundimonas (oral microbiome), Acetobacteraceae (skin microbiome), Afipia (unknown site, closest human microbiome homolog), and Ralstonia (gut microbiome). All of the bacterial homologs were also found to release citrulline during cleavage of glucosepane. Although the human homolog degrades glucosepane with similar activity to the bacterial homologs, citrulline was a minor degradation product, indicating that this homolog may function through a distinct mechanism. These are the first enzymes known to be capable of breaking free glucosepane, possibly making them useful starting points in the development of molecular tools to study glucosepane or as leads for therapeutic enzymes for AGE-associated diseases. The ability of these enzymes to cleave peptide bound glucosepane may have a variety of applications as therapeutics and research tools, including the potential use of these enzymes as a strategy to reverse the formation of glucosepane crosslinks within and between proteins to restore their native structure, an outcome that has significant therapeutic use. Example 2: Materials

N^e-(carboxyethyl)lysine (CEL) was purchased from Focus Synthesis, LLC. N^e- (carboxymethyl)lysine (CML) was purchased from Chem-Impex International. EZ-Tn5^TM <KAN-2>TNP Transposome^TM Kit was purchased from Epicentre. Ultra-micro (8.5 mm window height) cuvettes were purchased from BrandTech Scientific. Peptide Fmoc- D(otbu)E(otbu)FK(ivDDE)AD(otbu)E(otbu)-wang resin was synthesized by Biomatik. General experimental procedures

Low-resolution electrospray ionization-mass spectrometry (ESI-MS) data were collected on an Agilent 6120 Quadrupole LC/MS system equipped with a Phenomenex Kinetex C₁₈ (100 Å) 5-mm (4.6 x 250 mm) column. High-resolution (HR) ESI-MS data were obtained using an Agilent iFunnel 6550 QTOF MS instrument fitted with an electrospray ionization (ESI) source coupled to an Agilent 1290 Infinity HPLC system.

Spectrophotometric measurements were performed using a Thermo Scientific NanoDrop^TM 2000c Spectrophotometer. Strains

E. coli lysine auxotroph ( ^lysA) harboring kanamycin resistance gene (JW2806-1, Keio Collection) was obtained. However, transposomes in EZ-Tn5^TM <KAN-2>TNP Transposome^TM Kit contain the kanamycin resistance gene. Consequently, an E. coli lysine auxotroph strain sensitive to kanamycin was generated. To this end, the kanamycin resistance gene in JW2806-1 was excised through FLP recombination using the plasmid pCP20 encoding the FLP recombinase, which yielded a kanamycin-sensitive lysine auxotroph (JW2806-1_flpout) that was used as the host strain in the transposon mutagenesis studies. Sole lysine source growth studies, transposon mutagenesis and screening

To evaluate whether E. coli can grow in presence of CEL or CML as sole lysine source, E. coli strain JW2806-1_flpout was cultivated in 0.5 mL M9 minimal medium supplemented with 1 mg/mL lysine, 0.1% CEL, or 0.1% CML at 30 ^oC overnight under aerobic conditions. The same E. coli strain was then used as a host to generate a transposon mutant library using the EZ-Tn5^TM <KAN-2>TNP Transposome^TM Kit following manufacturer’s protocol. Briefly, electrocompetent JW2806-1_flpout cells were electroporated using 1 mL of the EZ-Tn5 <KAN-2> TNP Transposome.1 mL of SOC medium was then added to the cells and incubated on a 37 ^oC shaker for 1 hour to facilitate cell outgrowth. Dilutions of the aliquots of the recovered cells were then plated on M9 minimal medium agar plates supplemented with 50 mg/mL kanamycin and 1 mg/mL lysine. Resulting colonies were then picked into 96-well plates containing 300 mL of M9 minimal medium supplemented with 0.1% CEL and 50 mg/mL kanamycin, and cultured at 30 ^oC overnight under aerobic conditions. Genomic DNA from transposon mutants that showed impaired growth were then extracted using the DNeasy Blood and Tissue Kit (Qiagen), which was used as template for arbitrarily primed PCR. The amplified DNA segments near the transposon insertion sites were sequenced and subjected to BLAST analysis to identify the disrupted gene. MnmC expression and purification

N-terminally His₆-tagged MnmC was cloned and expressed as previously reported with slight modifications. In brief, DNA encoding MnmC was amplified from E. coli BW25113 genomic DNA with primers MnmC-fw and MnmC-rv (Table S1), which contain an NdeI site at the 5 ^-end and a BglII site at the 3 ^-end. Upon gel extraction (NucleoSpin Gel and PCR Clean-up, Macherey Nagel), the amplified sequence was cloned into pET28a (Novagen) expression vector between NdeI and BamHI sites. N-terminally His₆-tagged MnmC was then expressed in E. coli BL21 (DE3) (1L of TB media), grown to an OD₆₀₀ of 0.5 at 37 ^oC, induced with 0.4mM isopropyl b-D-1-thiogalactopyranoside, and further cultivated under aerobic conditions (250 rpm) overnight at 20 ^oC. Cells were lysed by sonication, and protein was purified from cell lysate using affinity chromatography (Ni-NTA agarose, Qiagen). Purified protein was then concentrated using an Amicon Ultra-15 centrifugal filter unit with 50 kDa cutoff, and prepared into glycerol stocks for storage at -20 ^oC. To clone N-terminally His₆-tagged C-MnmC (Pro251 to stop codon), primers C-MnmC- fw and C-MnmC-rv (Table S1) were used to amplify DNA from E. coli BW25113 genomic DNA, protein was isolated using identical purification steps as in MnmC before

concentrating with Amicon Ultra-15 centrifugal filter unit with 30 kDa cutoff. Site-directed mutagenesis

MnmC mutants were generated via the QuickChange mutagenesis method with the corresponding pairs of primers. pET28a construct containing MnmC gene was used as a template. All mutations were validated by sequencing. Hydrogen peroxide consumption assay

Hydrogen peroxide consumption by MnmC was monitored using a

spectrophotometric hydrogen peroxide assay as previously described. A typical experiment involved addition of H₂O₂ (40 mM) to MnmC (10 mM or 20 mM) or C-MnmC (10 mM) in a total volume of 200 mL in phosphate buffer (0.2 M, pH 7.6) and incubation at 37 ^oC for 1 h. 40 mL of chromogenic solution (1 mM vanillic acid, 0.5 mM 4-aminoantipyrine, 4 U/mL peroxidase in phosphate buffer) was then added to the mixture, and incubated at 37 ^oC for 20 min for color development before measuring the absorbance at 490 nm. Cleavage assay for CEL and DKP-CEL

In a typical cleavage assay, CEL or DKP-CEL (1 mM– 10 mM) was reacted with MnmC (1 mM– 5 mM) in PBS (pH 7.4) supplemented with FAD (5 mM) in a total volume of 100 mL for 2-3 h at 37 ^oC.50 mL of 2,4-DNPH solution (1 mM in 1 N HCl) was then added to the mixture, incubated at 37 ^oC for 10 min, basified with 350 mL of 0.6 N NaOH for color development, and the absorbance of the red-brown mixture at 445 nm was measured.

Calibration curve was obtained using pyruvate, which allowed determination of the amount of pyruvate generated after cleavage of CEL or DKP-CEL. Cleavage assay for Pept-CEL

Pept-CEL (16 mM) was reacted with C-MnmC (40 mM) in Tris buffer (50 mM, pH 8.0) supplemented with FAD (20 mM) in a total volume of 100 mL for 18 h at rt. The reaction mixture was blow-dried with nitrogen gas, and then extracted using 200 mL of MeOH before being subjected to LC-MS and HRMS/MS analysis. Cleavage assay for CML

CML (100 mM) was reacted with C-MnmC (25 mM) in Tris buffer (50 mM, pH 8.0) supplemented with FAD (20 mM) in a total volume of 50 mL for 4 h at 37 ^oC.25 mL of 2,4- DNPH solution (1 mM in 1N HCl) was then added to the mixture, incubated at 37 ^oC for 10 min, basified with 175 mL of 0.6 N NaOH for color development, and the absorbance of the orange mixture at 455 nm was measured. Calibration curve was obtained using glyoxylic acid, which allowed determination of the amount of glyoxylic acid generated after cleavage of CML. Enzyme kinetics

Reaction mixtures (50 mL) consisted of 50 mM Tris buffer (pH 8.0), 25 mM enzyme, 5 mM FAD, and CEL at various concentrations (1– 13 mM). All reactions were incubated at 37 ^oC, and stopped after 1– 21 min by adding 25 mL of 2,4-DNPH solution.175 mL of 0.6 N NaOH was then added to each reaction and the absorbance at 445 nm was measured. K_m and K_cat were determined from Michaelis-Menten plots using GraphPad Prism 7.

Synthesis of DKP-CEL and Pept-CEL

(1a). Boc-Lys(Z)-OSu (1.64 g, 3.4 mmol) and glycine methyl ester hydrochloride (453 mg, 3.6 mmol) were added to dichloromethane (70 mL). To the mixture, DIPEA (595 mL, 3.4 mmol) was added and the reaction was stirred at room temperature overnight, at which point a salt had formed. The solid was filtered off, rinsed with dichloromethane. The filtrate was washed with NaHCO₃ three times, dried over Na₂SO_4, and the solvent was removed under reduced pressure to yield 1a (1.35 g, 88 %).

(1b). To an ice-cold solution of 1a (1.35 g, 3 mmol) in dichloromethane (7 mL), TFA (7 mL) was added dropwise. Ice-bath was removed, at which point the reaction was warmed to room temperature and stirred for 1 h before the solvent was removed under reduced pressure to yield yellow oil. This product was dissolved back in butanol (20 mL), to which acetic acid (1 mL) and triethylamine (2 mL) were added and refluxed for 3 h at 120 ^oC. Upon cooling to room temperature overnight, crystals formed, which was cooled in ice-bath to complete crystallization and filtered off to yield 1b as white powder (287 mg, 30 %).

(1c). To a degassed solution of 1b (128 mg, 0.4 mmol) in dichloromethane (3 mL), acetic acid (3 mL) and Pd/C (10 wt-%, 36.5 mg) were added. H₂ was applied to the mixture, and was stirred at room temperature overnight. The reaction was then poured onto celite pad (~2 cm) on a glass frit, at which point the product was filtered through using methanol (~20 mL). The filtrate was then evaporated under reduced pressure to yield 1c as white powder (67 mg, 90%).

(1d).1c (60 mg, 0.32 mmol) was dissolved in 25% methanol/dichloromethane (8 mL) solution, to which trimethylamine (82 mL, 0.59 mmol) was added and let stir for 5 min. Ethyl pyruvate (0.3 mmol, 34 mL) was then added to the reaction as a solution in 25% methanol/ dichloromethane (4 mL). The reaction was stirred overnight at room temperature, after which the solvent was removed under reduced pressure and the product was resuspended in 50% methanol/dichloromethane on ice. Sodium borohydride (10 equiv.) was added in two portions 15 min. apart, and the reaction was stirred at room temperature overnight. Solvent was then removed under reduced pressure, at which point the resulting product was purified on Biotage using C18 column to yield mixtures of 1d and 1. Fractions containing these products were combined, further hydrolyzed by the addition of a few drops of 1 M lithium hydroxide solution, and HPLC-purified to yield 1 (3 mg).

Alkylation of lysine side chain

Pept-CEL was synthesized as reported previously with minor changes (4).2a (200 mg, 0.1 mmol) was swelled in DMF (2 mL) for 30 min. It was then agitated in 20% piperidine/DMF solution (2 mL, 2x) for 7 min, which yielded 2b. After thorough washing with DMF, 2b was then reacted with Boc₂O (20 equiv.) and DIPEA (10 equiv.) in DMF overnight which yielded 2c. To selectively deprotect lysine side chain, 2c was agitated in 4% hydrazine/DMF solution (2 mL, 3x) to yield 2d (quantitative). Small amount of 2d was cleaved from resin using TFA (1 mL) and subjected to LC-MS analysis to make sure there is no unreacted material. To alkylate lysine side chain, 2d (200mg, 0.13 mmol) was suspended in 1.5 mL DCM, to which pyruvate (14 mL, 0.2 mmol) was added and stirred for 5 min before adding sodium cyanoborohydride (15 mg, 0.24 mmol) dissolved in methanol (800 mL) and stirring overnight at room temperature. After global deprotection and cleavage from the resin using TFA/TIS/H₂O (950/25/25), 2 was precipitated in cold ether and filtered before being subjected to HPLC purification. Identification of a CEL-Breaking Biocatalyst

To assess whether E. coli was capable of cleaving the N-C bond in CEL or CML to restore Lys, free CML or CEL was fed as a sole Lys source to an E. coli Lys auxotroph ( ^lysA). E. coli ^lysA was cultivated in minimal M9 medium supplemented with 1 mg/mL Lys (positive control), CML, CEL or water vehicle (negative control) at 30 ^oC overnight under aerobic conditions. Strong growth was observed only for cultures supplemented with either Lys or CEL, indicating that E. coli can utilize CEL as a sole Lys source (FIG.4A). To identify the biocatalyst(s) responsible for CEL cleavage, transposon mutagenesis was performed in E. coli ^lysA and screened for mutants with impaired growth using CEL as a source of Lys (FIG.4B). One mutant from the non-saturating transposon mutant library (~3000 mutants were analyzed) exhibited impaired growth. The transposon insertion site was in mnmG (a.k.a., gidA), a gene located in the bacterial MnmEG pathway responsible for wobble U₃₄ tRNA modifications (FIG.4C). However, isolated MnmG was not able to cleave CEL in vitro (data not shown). Analysis of the broader pathway led to further study of MnmC, an enzyme that catalyzes a tRNA demodification reaction analogous to CEL cleavage (FIG.5A). Characterization of MnmC and validation of its activity on CEL

MnmC is a bifunctional enzyme that is known to catalyze two steps in the biosynthesis of the 5-methylaminomethyl-2-thiouridine (mnm⁵s²U₃₄) nucleoside in tRNA (FIG.5A). The C-terminal domain (C-MnmC) is an FAD-dependent oxidase that catalyzes demodification of the carboxymethyl functionality in cmnm⁵U₃₄ to generate a primary amine, and the N-terminal domain (N-MnmC) is a SAM-dependent methyltransferase that catalyzes methylation of this primary amine in nm⁵U₃₄ to yield mnm⁵U₃₄. These domains are fused by an interdomain linker which, when cleaved, yields the two domains in isolation that still retain catalytic activity. The C-MnmC domain-catalyzed demodification of a carboxymethyl functionality to a primary amine in a macromolecular target is analogous to the removal of a carboxyethyl (or carboxymethyl) functionality in the desired CEL-AGE (or CML-AGE) cleavage reaction (FIG.5B). D-amino acid oxidases (DAAOs) are the closest structural homologs of C-MnmC. Because DAAOs are FAD-dependent enzymes known to catalyze the oxidative deamination of various amines and neutral D-amino acids (e.g., glycine, sarcosine, D-alanine and D-proline) to give their corresponding a-keto acids, ammonia, and hydrogen peroxide (FIG.6A), MnmC’s ability to turnover CEL using an established spectrophotometric hydrogen peroxide assay was assessed using horseradish peroxidase (HRP) and chromogenic substrates. Like DAAOs, it was expected that MnmC-mediated cleavage of CEL could involve oxidation of the C-N^e bond to generate an imine, which is then hydrolyzed to yield pyruvate, L-Lys, and hydrogen peroxide (FIG.6B). However, hydrogen peroxide production was not detected, as this product appeared to be consumed by MnmC (FIG.11). Thus, to demonstrate that MnmC exhibits activity toward CEL, an a-keto-acid derivatization assay which can be used to monitor pyruvate formation as a result of CEL cleavage (FIG.6B) was then adapted. In short, 2,4-dinitrophenylhydrazine (2,4-DNPH) readily reacts with pyruvate to produce the 2,4-DNPH-pyruvate hydrazone adduct which has a distinctive red-brown color and absorbance maximum at 445 nm that can be monitored to measure the activity of MnmC on CEL (FIG.6C). At initial CEL concentrations of 1 mM and 10 mM, formation of 2,4- DNPH-pyruvate hydrazone adduct was detected in an MnmC-dependent manner. Formation of its 2,4-DNPH-pyruvate hydrazone products (syn- and anti- isomers) were confirmed by LC-MS using authentic standards (FIG.6D).

In addition to CEL, it was also tested whether MnmC is active on CML, which bears the carboxymethyl functionality similar to its native tRNA substrate. Despite the

resemblance, MnmC activity on CML was not observed at a 10 mM concentration, presumably due to differences in nonstandard substrate binding. This result is consistent with the observation that the E. coli Lys auxotroph does not grow in the presence of CML as a sole Lys source. Consistent with the overall findings, weak CML turnover using an MnmC variant was observed at higher substrate concentrations (100 mM, see below). Structure-based engineering of MnmC

To enhance the activity of MnmC on CEL, the X-ray crystal structure of E. coli MnmC (PDB: 3AWI) was evaluated. A cmnm⁵U₃₄ binding model of MnmC was generated, and 10 residues were identified around the bound substrate that can be mutated to better accommodate the longer hydrophobic alkyl chain of CEL (FIG.12). Relative activities of site-directed mutants at these positions were then compared using the a-keto acid derivatization assay (10 mM CEL, 5 mM MnmC, 4 hours at 37 ^oC). Three single residue mutants (Y312W, Y521L, and Y504K) showed only about a 2-fold increase in relative activities, and combinatorial double mutants did not show further improvements (FIG.7A). Since the reactions catalyzed by C-MnmC and N-MnmC are thought to be kinetically tuned, dissection and analysis of the desired C-terminal monodomain could exhibit improved properties. Thus, the N-terminal domain was cleaved off using a cut site within the interdomain linker (between Leu250 and Pro251), allowing isolated C-MnmC to be expressed in a catalytically competent form. Compared to the previously isolated C-MnmC, this C-MnmC variant contains an N-terminal His₆-tag with a shorter linker. This isolated C- MnmC monodomain construct exhibited about a 5-fold enhancement in relative activity compared to the wildtype didomain MnmC (FIG.7A), suggesting that the N-terminal domain could participate in quality control. In addition to E. coli MnmC, the activities of didomain MnmC homologs from related Gammaproteobacteria, Photorhabdus asymbiotica, Vibrio cholera, Photorhabdus luminescens, and Xenorhabdus bovienii, were also tested and their activities indicate that MnmC variants could more broadly catalyze CEL cleavage (FIG.7A).

Following the relative analyses of MnmC variants, the steady-state kinetics of wildtype didomain MnmC and monodomain C-MnmC were established for CEL using the a- keto acid derivatization assay (FIG.7B) and a 2,4-DNPH-pyruvate standard curve (FIG.13). The K_m values for MnmC and C-MnmC were comparable at 5.2 േ 1.3 mM and 4.3 േ 0.6 mM, respectively, whereas k_cat values were 0.04 േ 0.004 min^-1 and 0.18 േ 0.009 min^-1, respectively. These data indicate that the ~4.5-fold improvement of C-MnmC is largely a function of k_cat. Table 1 summarizes the kinetic parameters for CEL cleavage by MnmC and C-MnmC. Table 1. Kinetic parameters for CEL cleavage by MnmC and C-MnmC.

With the improved C-MnmC monodomain contruct, CML was evaluated as a substrate. CML turnover was only observed at a higher substrate concentration (100 mM CML, 20 mM C-MnmC, 4 hours, 37 ^oC). C-MnmC-catalyzed CML cleavage leads to glyoxylic acid and lysine (FIG.7C; FIG.14). Spectrophotometric detection of 2,4-DNPH- glyoxylic acid hydrazone adduct was monitored at 455 nm (FIG.7C) and quantified using a 2,4-DNPH-glyoxylic acid standard curve (FIG.7D; FIG.15). Substrate scope of C-MnmC

To have utility in probing the biological roles of CEL and CML AGEs, reversal biocatalysts must function on CEL/CML-modified peptide substrates. Because MnmC natively uses a macromolecular substrate like mature AGEs, the crystal structures of E. coli MnmC versus human DAAO were further evaluated. Superposition of C-MnmC and DAAO shows a high degree of structural similarity (FIG.8A). However, when the surface representations of the active sites for the two enzymes are compared, it becomes apparent that C-MnmC exhibits a wide and exposed active site whereas DAAO has a closed active site (FIG.8B and 8C). This difference likely arises from the fact that MnmC acts on a macromolecular tRNA substrate, whereas DAAO acts on small substrates (e.g., D-amino acids). The open active site in C-MnmC suggests that the enzyme could also utilize CEL in peptide contexts.

To investigate the substrate scope of C-MnmC, a CEL-modified diketopiperazine- CEL (DKP-CEL) was synthesized as a peptidomimetic of a mature CEL AGE (FIG.9A). Diketopiperazines are cyclic dipeptides that mimic protein beta turns. Indeed, C-MnmC was able to turn over peptidomimetic DKP-CEL using a 10-mM substrate concentration (FIG. 9B), suggesting that C-MnmC may cleave mature CEL residues. Activity for DKP-CEL was ~15% relative to free CEL. C-MnmC was tested for its activity in a peptide context by synthesizing a short linear peptide substrate DEF-(CEL)-ADE (Pept-CEL). Pept-CEL is a mature CEL-modified peptide (FIG.9C) that is known to bind to the positively charged cavity of the V domain of RAGE largely through its interaction with the carboxyethyl functionality. To detect activity of C-MnmC on Pept-CEL, an overnight enzymatic reaction containing 16 mM peptide substrate and 40 mM C-MnmC was analyzed by LC-MS. Pept- CEL was modestly converted to DEF-K-ADE in the presence of C-MnmC (FIG.6C).

HRMS/MS experiments were performed to sequence the substrate and product peptides to validate C-MnmC-mediated CEL to Lys residue cleavage (FIG.10A and 10B). These studies identified C-MnmC as the first catalyst capable of reversing a mature AGE modification and suggest that C-MnmC orthologs could serve as leads for further development.

Despite numerous studies establishing AGEs as contributing factors to the progression of degenerative diseases, it has been a challenge to establish causal relationships between them. One major challenge is an inability to reverse AGE modifications and evaluate potential disease amelioration. Biocatalysts that are capable of demodifying specific mature AGEs can lead to a new arsenal of molecular tools for the study of AGEs, and to recombinant-enzyme therapies. However, no such tools exist. Here, it was found that an E. coli lysine auxotroph can utilize CEL as a sole lysine source. Through a combination of transposon mutagenesis, screening, synthesis, and protein biochemical studies, MnmC was identified and characterized as an enzyme capable of CEL cleavage and Lys restoration. While the non-saturating transposon studies initially led to the identification of mnmG, it could not be shown that MnmG-mediated CEL cleavage. This prompted examination of the broader bacterial MnmEG pathway. Previous reports on the MnmEG pathway established that it is tightly controlled, suggesting the possibility that mnmG disruption could affect other genes in the pathway. This led to focus on MnmC, which catalyzes an analogous reaction to CEL demodification.

Previous homology searches on MnmC revealed that its C-terminal domain is structurally related to glycine oxidases (GOXs), sarcosine oxidases (SOXs), and DAAOs. As FAD-dependent oxidases, these enzymes catalyze a similar enzymatic reaction where an N-C bond is oxidized to an imine which is subsequently hydrolyzed. Intriguingly, C-MnmC is known to catalyze a reaction in which imine generation followed by hydrolysis results in a decarboxymethylation to release a primary amine. Homology modeling efforts showed that C-MnmC is structurally closest to the DAAOs. High structural homology between C-MnmC and DAAO could explain the fact that CEL, which is in effect an N-alkyl alanine, is a better substrate than CML, which is in effect an N-alkyl glycine. DAAOs are known to accept glycine as a substrate, albeit with much lower affinity than functionalized D-amino acids (Table 2). This is consistent with the observation that C-MnmC, as a homolog of DAAOs, shows much weaker activity toward CML than CEL.

Table 2:

During the course of these studies, hydrogen peroxide scavenging activities by both full-length MnmC and C-MnmC were unexpectedly observed (Fig.11). The crystal structure of E. coli MnmC (PDB: 3PS9) shows a chloride ion near the isoalloxazine ring of FAD. This resembles the presence of a chloride ion near the FAD in RebH (PDB: 2OA1) (FIG.16), an FAD-dependent halogenase in which peroxy-flavin is attacked by the chloride ion to generate the chlorinating agent HOCl instead of hydrogen peroxide. These similarities might explain hydrogen peroxide quenching by MnmC.

The capability of C-MnmC to demodify CEL in free, peptidomimetic, and mature peptide contexts can likely be attributed to the enzyme’s open and large active site. Similarly, Amadoriases I and II, which are well-known FAD-dependent oxidases capable of oxidizing the early Amadori product (e.g., fructoselysine), are also known to be functional on peptide substrates with similar K_m values (4.2 mM and 5.6 mM for glycated-L-Lys and glycated-Lys- Phe, respectively). Upon examination of the active sites of these Amadoriases, it becomes apparent that their active sites are also quite large, which could explain how these enzymes are able to function on peptide substrates unlike DAAOs and GOXs with small, buried active sites (FIG.17). One exception is the DAAO from the yeast Rhodotorula gracilis (RgDAAO). With an unusually large active site, the RgDAAO is known to accept cephalosporin C and convert it to glutaryl-7-aminocephalosporanic acid, a key intermediate in the enzyme- catalyzed synthesis of cephalosporin antibiotics. CEL demodification and cephalosporin C inactivation mediated by these types of enzymes represent alternative activities that could be exploited for non-canonical functions.

In summary, this study has shown that C-MnmC is a biocatalyst that can cleave mature CEL modifications to restore native lysine structures. This is the first biochemical characterization of an enzyme that can demodify a mature AGE-functionalized peptide. While the kinetic parameters, which are similar to known Amadoriases, could be

substantially improved, C-MnmC variants represent lead catalysts for further development. Such improved AGE-reversal tools, in certain embodiments, enable a better understanding of the biology of AGEs at the molecular level, elucidate their direct roles in the pathogenesis of age-related diseases, and serve as recombinant enzyme-therapy agents.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

What is claimed is: 1. A method of removing glucosepane from a subject’s tissue, the method comprising administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a biologically active fragment thereof.

2. A method of treating or preventing an advanced glycation end-product associated (AGE-associated) disease in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a biologically active fragment thereof.

3. The method of claim 2, wherein the AGE-associated disease comprises Alzheimer’s disease, diabetes, atherosclerosis, joint stiffening, skin wrinkling, and//or thickening of basement membranes in capillaries, glomeruli, lens, and/or lungs.

4. The method of any of claims 1-3, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-11.

5. The method of any of claims 1-4, wherein the polypeptide further comprises at least one selected from the group consisting of a cell-penetrating peptide, a cell secretion signaling peptide, and a stability enhancing domain.

6. The method of any of claims 1-5, wherein the polypeptide is formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

7. The method of claim 6, wherein the composition is formulated for topical or ocular administration.

8. The method of any of claims 1-7, wherein the polypeptide is administered topically or ocularly to the subject.

9. The method of any of claims 1-8, wherein the subject is a mammal.

10. The method of any of claims 1-9, wherein the subject is a human.

11. A method of treating or preventing an advanced glycation end-product associated (AGE-associated) disease in a subject, the method comprising administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13, or a biologically active fragment thereof.

12. A method of removing N^e-(carboxyethyl)lysine (CEL) or N^e-(carboxymethyl)lysine (CML) from a subject’s tissue, the method comprising administering to the subject an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-13, or a biologically active fragment thereof.

13. The method of any of claims 11-12, wherein the polypeptide further comprises at least one selected from the group consisting of a cell-penetrating peptide, a cell secretion signaling peptide, and a stability enhancing domain.

14. The method of any of claims 11-13, wherein the polypeptide is formulated as a pharmaceutical composition further comprising at least one pharmaceutically acceptable excipient.

15. The method of claim 14, wherein the composition is formulated for topical or ocular administration.

16. The method of any of claims 11-15, wherein the polypeptide is administered topically or ocularly to the subject.

17. The method of any of claims 1, 4-10, and 12-16, wherein the tissue comprises an extracellular matrix (ECM).

18. The method of any of claims 1, 4-10, and 12-17, wherein the tissue comprises a basement membrane of the ECM, joint tissue, and/or skin.

19. The method of any of claims 11-18, wherein the subject is a mammal.

20. The method of any of claims 11-19, wherein the subject is a human.

21. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, or a biologically active fragment thereof, wherein the composition is formulated for topical or ocular administration.