WO2015073538A2 - Interaction with metalloenzymes - Google Patents

Abstract

Compositions comprising peptides that are capable of inhibiting metalloenzymes are disclosed. The peptides can be used for the treatment of cancers associated with metalloenzymes.

Description

INTERACTION WITH METALLOENZYMES

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No.

61/903,303, filed on November 12, 2013, the contents of which are incorporated by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Nos. NIH

RO1DE014392 and Grant Nos. RO1DE022054 awarded from the National Institute of Health. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Peptide tags that can be encoded in the genetic material of an organism for recombinant expression of proteins have been utilized for purification and identification of protein products. The advantage of a peptide tag is that the tag is covalently attached to the protein of interest without the need for additional chemical steps to label the protein. Peptide- based tags have been developed to allow for detecting a tagged protein in cell culture assays or cell lysates using antibodies that recognize the peptide tag.

[0004] While these technologies might be useful in in situ or in vitro assays, they generally are not applicable to in vivo analysis. Moreover, such peptide tags have limited or no functionality outside of protein purification or identification.

[0005] Matrix metalloproteinases (MMP) are a family of zinc-containing metalloenzymes that have the ability to degrade components of the extracellular matrix and basement membranes. Expression of matrix metalloproteinases increases in various pathological conditions such as tumor growth and metastasis. MMP-based inhibition strategies have therapeutic potential for the treatment of cancer.

SUMMARY OF THE INVENTION

[0006] In some embodiments, the invention provides a method of inhibiting a

metalloenzyme in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a composition comprising a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[0007] In some embodiments, the invention provides a method of treating a cancer associated with a metalloenzyme in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a composition comprising a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[0008] In some embodiments, the invention provides a method of inhibiting a

metalloenzyme, the method comprising contacting the metalloenzyme with a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGURE 1 illustrates a schematic representation of a MAP peptide- grafted polymer surface to inhibit MMP-8. Panel a depicts amine-terminated polymer surfaces. Panel b depicts grafting of tether-MAP peptide (SEQ ID No. 1) to amines via DSS linker chemistry. Panel c depicts MMP-8 inhibition at polymer surface by MAP peptide NCC. The ribbon structure of MMP-8 was derived from protein data bank (PDB) entry 20Y4.

[0011] FIGURE 2 illustrates the reaction and product of the experiments of Example 9. A bar graph shows the percent activity remaining at 30-minute endpoint comparison from the MMP-8 fluorometric RED assay with increasing concentrations of MAP peptide NCC and tether-MAP (SEQ ID No. 1). The standard inhibitor NNGH was used as a control. The bar graph shows that MAP peptides inhibited MMP-8 activity in solution.

[0012] FIGURE 3 illustrates the reaction and product of the experiment of Example 6 and Example 7. Accessibility of amine functionality and grafting of peptide onto amine-doped polymer surfaces is shown. The Y-axis in Panel a reflects methyl orange absorbance at 465 nm for the control (0%), 5%, 10%, 15%, and 20% amine-containing polymer samples before and after DSS coupling. The Y-axes in Panel b shows fluorescence intensity of grafted polymers in RFU. The left axis corresponds to the intrinsic fluorescence intensity (E_ex/E_em= 280/360 nm) of the single tryptophan residue in tether-MAP (SEQ ID No. 1), and the right y- axis corresponds to the fluorescence intensity of grafted FITC (E_ex/E_em= 492/516 nm). The figure illustrates effective coupling to polymer-incorporated amines was accomplished.

[0013] FIGURE 4 illustrates the reaction and product of the experiments of Example 10. The bar graph illustrates the results of MMP-8 fluorimetric RED assay carried out on control (0%), 5%, 10%, 15%, and 20% amine-containing polymers grafted with tether-MAP (SEQ ID No. 1). The standard inhibitor NNGH was used as a control. The graph shows that MMP- 8 activity was inhibited by MAP-grafted polymer surfaces.

DETAILED DESCRIPTION

[0014] Metalloenzymes are implicated in various biological processes. Although the structures, functions, and metal binding sites exhibit great diversity, the commonality is that the activity of the metalloenzyme is predicated upon hosting a metal. In many cases, the metal is functional and participates in catalysis, such as by binding a substrate, promoting electron transfer, creating a hard or soft site, creating a nucleophilic or electrophilic site, organizing guests within a binding site, or otherwise regulating the local environment. The metal can also be structural, not participating in catalysis, but providing a superstructure or conformation that is beneficial or necessary for catalysis. Metalloenzymes frequently bind a metal specifically, at a particular site using particular coordinating groups, which can be selective for particular metals in particular oxidation states, electronic configurations, or coordination geometries. In some cases, a metal is bound non- specifically and is still relevant to a metalloenzyme' s physiological function.

[0015] Modulation of the metalloenzyme/metal complex can affect the enzymatic function, and interference with the complex can attenuate function. An agent, such as the metal abstraction peptide (MAP) described herein, can modulate or interfere with such complexes. Non-limiting examples of mechanisms to interfere with the enzymatic function include removing the metal from the metalloenzyme, disrupting or weakening the host/guest complex, replacing the metal with one that binds more weakly to the metalloenzyme, competing for binding with the complexed metal, or establishing an equilibrium for metal biding with the metalloenzyme versus another agent.

[0016] Another agent can interfere with the metalloenzyme/metal complex by simultaneously binding the complexed metal and blocking the metal site on the metalloenzyme. Possible results are occluding the entry of substrates or co-enzymes, disrupting a conformation of the metalloenzyme, modifying the metal's coordination sphere, electron configuration, or binding geometry, and poisoning or doping the metalloenzyme reversibly or irreversibly. Binding can be selective or non-selective across enzyme types or subtypes.

[0017] Non-limiting examples of metalloenzymes include hydrogenases, aconitases, glucose 6-phosphatases, DNA polymerases, hexokinases, arginases, ureases, hydratases, methionyl aminopeptidases, methylmalonyl-CoA mutases, isobutyryl-CoA mutases, laccases, cytochrome oxidases, dehydrogenases, alcohol dehydrogenases, carboxypeptidases, aminopeptidases, nitrate reductases, sulfite oxidases, xanthine oxidases, glutathione peroxidases, and matrix metalloproteinases.

[0018] In some embodiments, the present invention relates to the use of a peptide of the disclosure to inhibit a metalloenzyme. Inhibition of the metalloenzyme can reduce enzymatic activity of the metalloenzyme. The enzymatic activity of the metalloenzyme can be reduced by a peptide of the disclosure by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the metalloenzyme activity of a wild-type enzyme. The enzymatic activity of the metalloenzyme can be reduced by a peptide of the disclosure by at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, or at most about 100% compared to the enzymatic activity of a wild-type enzyme.

[0019] In some embodiments, a method is provided for treating a disease, for example, cancer, associated with a metalloenzyme in a subject in need thereof comprising

administering a peptide of the disclosure.

Matrix metalloproteinase (MMP).

[0020] Matrix metalloproteinases (MMPs) are a class of calcium-dependent zinc-containing endopeptidases capable of degrading essentially all components of the extracellular matrix (ECM) including collagens, elastins, gelatin, matrix glycoproteins, and proteoglycan. MMPs require a zinc ion in their active site for catalytic activity. MMPs play a critical role in physiological ECM remodeling such as during tissue morphogenesis, growth, tissue repair, and angiogenesis.

[0021] The MMP family is large and the members act on diverse substrates specific to individual tissues, for example, mature human odontoblasts secrete the gelatinases MMP-2 and -9, collagenases MMP-8 and -13, and enamelysin MMP-20. MMPs can be divided into subgroups based on structure and substrate specificity. The MMP family has over twenty members, including collagenases, gelatinases, stromelysins, elastases, matrilysins, aggrecanases, and membrane-bound MMPs (MT-MMPs).

[0022] MMPs have a multidomain structure comprising a conserved catalytic domain that incorporates a propeptide, an N-terminal signal peptide that directs secretion of pro-MMP from the cell, and a C-terminal hemopexin domain that contributes to substrate specificity and interaction with TIMPs. The propeptide comprises a highly conserved amino acid sequence. A covalent bond between the cysteine residue of the propeptide and a zinc ion of the catalytic domain is necessary to maintain the pro-MMP in latent form. The catalytic domain comprises two modules separated by a deep, active site cleft with catalytic zinc ion at the bottom. A zinc-binding motif comprising three histidine residues coordinates the binding of the catalytic zinc at the active site. The zinc-binding motif together with the catalytic zinc ion is essential for the proteolytic activity of MMPs and is conserved among all MMPs. MMPs also comprise a structural zinc and at least one calcium ion. The C-terminal hemopexin domain is linked to the catalytic domain by a proline-rich hinge region. The C- terminal hemopexin domain is highly conserved in MMPs and shows sequence similarity to hemopexin, a plasma protein. In addition to these domains, some MMPs can possess more domains.

[0023] Abnormal expression of MMP activity can lead to excessive degradation of ECM components. Non-limiting examples of pathological conditions associated with abnormal MMP activity include destruction of cartilage and bone in rheumatoid and osteoarthritis, degradation of myelin-basic protein in neuroinflammatory diseases, opening of the blood- brain barrier following brain injury, increased matrix turnover in restenotic lesions, loss of aortic wall strength in aneurysms, tissue degradation in gastric ulceration, skeletal dysplasia, liver fibrosis, acute lung injury, acute respiratory distress syndrome, autoimmune blistering disorder of the skin, dermal photoaging, breakdown of connective tissue in periodontal disease, and tissue breakdown and remodeling during invasive tumor growth and tumor angiogenesis in cancer.

Matrix metalloproteinase (MMP) role in cancer.

[0024] Overproduction of MMPs has been implicated in cancer, for example, during tumor growth, metastasis and cancer cell survival. Tumor growth can involve alterations in the ECM. Metastasis can involve detachment of the malignant cells from the primary tumor, invasion through the ECM, entry into circulation, invasion of the target organ, and formation of a metastatic colony. Tumor-induced angiogenesis is essential for growth of the primary tumor and metastases, and new blood vessels are sites for entry of tumor cell entry into the circulation.

[0025] Malignant tumors are often characterized by increased expression of MMPs relative to normal tissue. High levels of expression of MMPs can be associated with a poor prognosis of the disease. MMPs can be induced by the cancer cell to reconstruct adjacent normal tissue to allow neovascularization, tumor growth and metastasis. MMPs can degrade the surrounding matrix tissue. Additional functions mediated by MMPs in cancer include activation of growth factors, suppression of tumor cell apoptosis, destruction of chemokine gradients developed by host immune response, and release of angiogenic factors.

Matrix metalloproteinase (MMP) inhibition.

[0026] The activity of MMP is regulated by a group of endogenous inhibitory proteins such as a2-macroglobulin, which is a plasma protein that acts as general protease inhibitor, and a specific group of proteins called the tissue inhibitors of metalloproteinases (TIMPs). TIMPs can bind to active and alternative sites of the activated MMP. An imbalance between MMPs and MMP inhibitory proteins can allow the destruction of the extracellular matrix. In pathological conditions such as cancer, this destruction of the extracellular matrix by MMPs can enhance the ability of tumor cells to grow and/or metastasize. Consequently, MMP- inhibitory strategies can be used for the treatment of cancer. A MMP inhibitor can be used to restrict invasive tumor growth and/or metastasis. A MMP inhibitor can also provide anti- angiogenic properties to treat cancer.

[0027] The large size (about 20-30 kDa) of endogenous MMP inhibitors, for example, TIMPs, makes them impractical for therapeutic use in MMP-associated diseases. Synthetic inhibitors can be designed to inhibit MMPs. A synthetic MMP inhibitor can bind to and/or chelate a metal from any metal-binding site of the enzyme, thereby blocking the activity of the MMP. The inhibition can be specific for the known matrix metalloproteinase family and selective or unselective among family members. The inhibition can be reversible.

[0028] In some embodiments, the present invention relates to the use of various peptides to inhibit MMP activity. The MMP activity can be inhibited by the peptides of the disclosure by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the MMP activity of a wild-type enzyme. The MMP activity can be inhibited by the peptides of the disclosure by at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, or at most about 100% compared to the MMP- 8 activity of a wild- type enzyme. [0029] Matrix metalloproteinase-8 (MMP-8), also referred to as neutrophil collagenase or collagenase-8, is a member of the MMP subfamily collagenases. MMP8 can be expressed in a wide range of cells, including neutrophils, leukocytes, chondrocytes, epithelial cells, fibroblasts and macrophages. Inactive MMP-8 can be stored in intracellular granules of neutrophils and released in response to extracellular stimuli such as inflammation.

[0030] MMP-8 plays a role in various normal and pathological conditions due to a broad substrate specificity. Targets of MMP-8 can include collagen, gelatin, aggrecan, entactin, and al-proteinase inhibitor. MMP-8 is secreted as a 55-80 kDa glycosylated proenzyme, and activated by cleavage.

[0031] MMP-8 is an important target for inhibitor screening due to involvement in inflammation, wound healing and diseases such as multiple sclerosis, arthritis, and asthma. MMP-8 has also been implicated in cancer progression. MMP-8 can play an important role in the invasion process of cancer. Expression levels of MMP-8 can correlate with tumor stage and poor prognosis in cancer.

[0032] Non-limiting example of cancers associated with altered MMP-8 activity include melanoma, ovarian cancer, colorectal cancer, Waldenstrom's macroglobulinemia (cancer of B lymphocytes), head and neck cancer, lung cancer, hepatocellular carcinoma, breast cancer, superficial bladder cancer, gastric cancer, renal cancer, pancreatic cancer, neuroectodermal cancer, cervical cancer, prostate cancer, melanoma, and sarcoma.

[0033] In some embodiments, the present invention relates to the use of various peptides to inhibit MMP-8 activity. The MMP-8 activity can be inhibited by the peptides of the disclosure by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the MMP-8 activity of a wild-type enzyme. The MMP-8 activity can be inhibited by the peptides of the disclosure by at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, or at most about 100% compared to the MMP-8 activity of a wild-type enzyme.

Role of MMPs in dental reconstruction.

[0034] MMP activity remains a prime concern in the longevity of biocompatible polymers, including dental adhesives. The prevention and treatment of tooth decay are major challenges in dentistry and annual expenditures associated with dental services surpass $100 billion dollars in the US. A substantial portion of this economic burden arises from the need for replacement of dental reconstructions. Dental materials are softer and more porous than the mineralized tooth and as such leach bioactive molecules from dentin that activate key enzymatic reactions, particularly MMPs. These host-derived enzymes degrade the supporting tooth structure to which the adhesive is attached and facilitate destruction of the bonded interface.

[0035] In addition to MMP activity, a second key factor that can contribute to premature failure of composite restorations is recurrent caries at the margins of the dental restorations. This phenomenon is linked to attachment of the cariogenic bacterium Streptococcus mutans (S. mutans). Adhesion of S. mutans to the tooth surface supports subsequent growth and recruitment of additional bacteria. Microbial metabolism generates lactic acid, which demineralizes the tooth and further activates MMPs. Efforts to address bacterial adhesion and MMP activity have primarily relied on incorporation of a basic co-monomer. This approach is non-specific in the mechanism of action. In some embodiments, amine moieties are used to tether an inhibitory peptide to the methacrylate polymer to achieve specific inhibition.

[0036] Dentin is composed predominantly of type I collagen, and cleavage of this matrix is primarily accomplished by MMP-8. As such, inhibition of MMP-8 is significant in developing next-generation dental adhesives. In dental reconstruction, the release and subsequent activation of MMP-8 is responsible primarily for accelerating degradation of collagen fibrils in incompletely infiltrated aged bonded dentin hastening the need for replacement of reconstructions. Therefore, development of a dental adhesive that better resists MMP-8 activity is of significant interest. Herein, the metal abstraction peptide (MAP) is identified as an inhibitor of MMP-8 and demonstrate that tethering MAP to methacrylate polymers effectively inhibits catalysis. A complete inhibition of MMP-8 is achievable using a grafting approach. This strategy has potential to improve longevity of dental adhesives and other polymers and enable rational design of a new generation of biocompatible materials.

[0037] A fluorimetric activity assay was employed to examine MMP-8 inhibition by MAP peptides, including as a non-limiting example the base unit NCC. The MAP peptides were tested and compared with positive and negative control reactions. FIGURE 2 shows that 0.10 μΜ MAP peptide NCC results in nearly complete inhibition of MMP-8, as compared to 1.3 μΜ of the standard inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH), indicating the MAP peptide NCC effectively inhibits this enzyme.

[0038] In order to ensure accessibility of the inhibitory peptide and permit interaction with MMP-8 once tethered to the polymer, MAP peptide NCC was incorporated into a longer polypeptide to provide a spacer between the inhibitory MAP module NCC module and the polymer surface. Based on examination of the structures of TIMPs and structure activity relationships of MMP-8 inhibitors, a thirteen amino acid spacer was chosen which includes a tryptophan for quantification. A sixteen amino acid long peptide called tether- MAP (SEQ ID No. 1) was generated with MAP peptide NCC at the C-terminus. The tether-MAP (SEQ ID No. 1) peptide was tested for the ability to inhibit MMP-8 activity in solution and was found to completely abrogate turn over at comparable concentration to NNGH as illustrated in

FIGURE 2 panel b.

[0039] To facilitate covalent attachment of peptide to the polymer, methacrylate resin was doped with an amine-containing monomer and a homo-bifunctional amine-reactive linker was used for coupling. Before the tether-MAP inhibitor peptide (SEQ ID No. 1) was grafted onto the surface of modified polymer samples, the effect of the amine-containing monomer on polymerization was established.

[0040] The quality of the adhesive bond to the dentin substrate can be closely related to infiltration and photo-polymerization of adhesive resins. Polymerization was quantified using the standard FTIR methodology to measure degree of conversion (DC). DC value is a critical parameter that reflects the quality of the adhesive bond and the interfacial hybrid layer. The standard dental adhesive formulation currently used contains 45% hydroxyethyl methacrylate (HEMA) and 55% 2,2-bis[4-(2-hydroxy-3- methacryloxypropoxy)phenyl]- propane (BisGMA) and has a DC of 79%. Here, the formulation was modified to contain 60% HEMA, which was partially replaced with 2-aminoethyl methacrylate hydrochloride (AEMA). Samples containing 5%, 10%, 15% and 20% AEMA were polymerized. The results of this study indicated the DC of the experimental adhesives were higher than that of the control formulation, containing 60% HEMA and 40% BisGMA. The data shows DC increased from 61% for the control resin to 70-80% for the experimental resins. The observed increase in DC of AEMA-containing resins can be attributed to decreased viscosity resulting from the lower BisGMA content, which also can improve infiltration into the dentin substrate.

[0041] Polymerized samples containing AEMA should have solvent accessible amines. For confirmation, a methyl orange (MO) assay was used to determine the amount of solvent accessible amine for each polymer sample. MO binds to amines and is extracted effectively by incubation in basic solution. MO solutions extracted from polymer samples were quantified using absorbance spectroscopy at 465 nm Data indicated an increasing amount of amine was exposed with higher percent AEMA-containing polymer and that solvent accessible amine content increase in proportion to the concentration of AEMA in the resin, as expected (FIGURE 3).

[0042] Disuccinimidyl suberate (DSS) was used to link tether-MAP peptide (SEQ ID No. 1) to the polymer surface via the N-terminus. Peptide attachment was quantified using intrinsic tryptophan fluorescence and the residual unreacted amine content quantified using the MO assay. As a positive control for functionalization of the material, separately a fluorescence dye 4'-(aminomethyl) fluorescein, hydrochloride (FITC) was reacted with polymer samples for comparison. The grafting of both tether-MAP (SEQ ID No. 1) and FITC followed a nonlinear trend in fluorescence with increasing AEMA content in the polymer resin (FIGURE 3).

[0043] Following the grafting reaction, the methyl orange assay was performed to determine the amount of residual solvent accessible amines within the polymer. A comparison of the results from non-amine containing surfaces with grafted surfaces indicated that the grafting reaction was efficient and reproducible, as the total and residual amine content followed the same non-linear trend. No discernible difference was observed in the control formulation before and after DSS-dye coupling, but a decrease in average absorbance at 465 nm was observed for the 5%, 10%, 15%, and 20% amine polymers after DSS-dye coupling when compared with the results from samples prior to grafting (FIGURE 3). In all cases, MO binding decreased approximately 80% following the reaction, indicating -20% of the amine content remains available (FIGURE 3). This result suggests that incorporation of amine moieties can provide multi-functionality, in which the grafted peptide specifically staves off the destructive action of MMPs while unreacted amines act as a proton sponge to moderate the effects of chemical degradation caused by the acidic oral environment.

[0044] In both the fluorescence and methyl orange assays the concentration dependence appears to be non-linear with respect to the amine concentration in the resin, suggesting the solvent-accessible amine content is affected by chemical and/or structural changes in the polymer. The effect can emanate from variation in the formulation's overall hydrophobicity, which can perturb the pKa and/or reactivity of the amine differently in the context of resin mixtures having differing properties. Relative proportions of individual components can also affect surface accessibility of the amine groups and crosslinking density by modulating viscosity and/or miscibility. The results demonstrate increasing the proportion of amine- containing monomer in the formulation disproportionately elevates the solvent accessible amine content. Nonetheless, the ratio of reacted to residual amines remains constant.

[0045] To verify that the peptide remains functional as an inhibitor after tethering to the polymer surface, the MMP-8 assay was carried out on the series of peptide grafted polymer surfaces, including control resin, 5%, 10%, and 20% AEMA-doped resin. Each resin was first modified with tether-MAP (SEQ ID No. 1) and then assayed as in solution and compared with control and NNGH reactions. In addition, the control and standard inhibitor reactions were run on the series of unconjugated, bare polymer resins to account for background and establish any differences caused by changes in polymer composition. All resins behaved comparably, indicating the polymer does not affect the assay and that the observed inhibition results from the grafted peptide. FIGURE 4 shows the efficiency of inhibition increased with increasing amount of grafted peptide, corresponding to the percent amine in the formulation. The 20% AEMA-containing resin grafted with tether-MAP (SEQ ID No. 1) showed the best result, in which complete inhibition of MMP-8 was achieved (FIGURE 4).

[0046] The MAP peptide NCC effectively inhibited MMP-8 and the inhibitory function was retained when tethered to the polymer surface via a spacer. The standard dental adhesive resin formulation was modified to incorporate functionality for peptide grafting, and incorporation of AEMA produced polymers with the desired degree of conversion. An efficient method to graft peptide to the polymer and achieved complete inhibition of MMP-8 at the polymer surface. Optimization of the composition of the dental adhesive formulation to maximize performance characteristics clearly involves multiple parameters.

Metal Abstraction Peptide (MAP).

[0047] The present disclosure provides peptide motifs and methods of using such motifs. These peptides have the ability to bind to metals, which makes the resultant complexes useful for a variety of applications. Peptides of the present disclosure have applications in cancer therapy and modulation of formulation rheology.

[0048] The present disclosure provides a peptide comprising the sequence XCiC₂; wherein X is any natural or non-natural amino acid or amino acid analogue such that XQC₂ is capable of binding a metal. In some embodiments, the peptide is capable of binding metal in a square planar orientation, a square pyramidal orientation, or both. In some embodiments, Ci and C₂ are the same or different; and Ci and C₂ individually are sulfur containing amino acid residues such as cysteine and a cysteine-like non-natural amino acid or amino acid analogue. In some embodiments, Q and C₂ are each individually chosen from sulfur-containing alpha or beta amino acids.

[0049] The present disclosure also provides a peptide comprising the sequence XCiC₂ and a bound metal, wherein the metal is complexed with or bound to the tripeptide. In some embodiments, X is any natural or non-natural amino acid or amino acid analogue such that XC₁C₂ and the bound metal are in a square planar orientation, a square pyramidal orientation, or both, and wherein Q and C₂ are the same or different, and wherein Q and C₂ individually are sulfur containing amino acid residues such as cysteine and a cysteine-like non-natural amino acid or amino acid analogue. In some embodiments, Ci and C₂ are each individually chosen from sulfur-containing alpha or beta amino acids.

[0050] The present disclosure provides methods comprising complexing a metal together with a tripeptide having the sequence XC₁C₂ to form a metal-XCiC₂ complex, wherein X is any natural or non-natural amino acid or amino acid analogue such that metal-XCiC₂ complex has a square planar orientation, a square pyramidal orientation, or both, and wherein Ci and C₂ are the same or different, and wherein Q and C₂ individually are sulfur containing amino acid residues such as cysteine and a cysteine-like non-natural amino acid or amino acid analogue. In some embodiments, Ci and C₂ are each individually chosen from sulfur- containing alpha or beta amino acids. The X in the MAP sequence can be any natural or non- natural amino acid.

[0051] In some embodiments, the invention provides peptide motifs that strongly bind with a select metal. In some embodiments, the metal is a select metal, or a specific metal. For example, a peptide can have selectivity for biding one metal over another, or one oxidation state of a metal over another oxidation state of the same metal or a different metal. Such peptides are referred to as metal abstraction peptides (MAP(s)). MAPs can be used, for example, to bind a metal in a composition. MAPs can be included in longer polypeptides and proteins at the N-terminus, C-terminus, or any position in between. In some embodiments, a MAP can be present in a polypeptide or protein configuration that presents the MAP for binding with a metal, such as being present in an external loop. The MAP can be covalently attached to a polypeptide or protein through a linker, such as at the N-terminus, C-terminus, or through a side-chain from the polypeptide or protein. For example, such linkers can include amide bonds, esters, polyamides, polyethers, and polyesters. The MAP can be attached to a non-peptide entity. Non-peptide entities include without limitation

carbohydrates, glycoproteins, and/or covalent linkers, including polyethylene glycol.

Additionally, more than one MAP can be present on a particular molecule. In some embodiments, one or more MAPs can be covalently linked to an antibody. In some embodiments, the MAP is a tripeptide capable of complexation with metal ions, as described in U.S. Patent Publication 2010/0221839. Chemical Structure/Pep tide Sequence.

[0052] As used herein, the abbreviations for the L-enantiomeric and D-enantiomeric amino acids are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine (Q, Gin); glycine (G, Gly); histidine (H, His); isoleucine (I, He); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Typically, X or Xaa can indicate any amino acid. However, in some embodiments, X or Xaa is selected from a subset of amino acids. In some embodiments, the amino acid is a L-enantiomer. In some embodiments, the amino acid is a D-enantiomer.

[0053] In some embodiments, XC₁C₂ are L-amino acids or are achiral, such as glycine. Non- limiting examples of X include hydrophilic amino acids, hydrophobic amino acids, charged amino acids, uncharged amino acids, acidic amino acids, basic amino acids, neutral amino acids, aromatic amino acids, aliphatic amino acids, natural amino acids, and non-natural amino acids. In some embodiments, X is selected from N, Q, H, K, and R. In some embodiments, XCiC₂ are L-amino acids, and X is selected from N, Q, H, K, and R. In some embodiments, X is achiral, for example, X is glycine. In some embodiments, X is not N, Q,

H, K, R, or G. In some embodiments, X is not cysteine. In some embodiments, X is selected from alanine (A); aspartic acid (D); glutamic acid (E); isoleucine (I); leucine (L);

methionine (M); phenylalanine (F); serine (S); threonine (T); tryptophan (W); tyrosine (Y); and valine (V).

[0054] A MAP tag can have a net charge of -5, -4, -3, -2, -1, 0, +1, +2, +3, +4, or +5. When the net charge is zero, the MAP can be uncharged or zwitterionic. Addition of a metal to a MAP can change the net charge of the complex, for example, by decreasing the net charge by

I, 2, 3, 4, or 5, or by increasing the net charge by 1, 2, 3, 4, or 5. In some embodiments, addition of a metal to a MAP does not change the net charge of the complex.

[0055] In some embodiments, the MAP tag can be attached to another molecule. For example, the MAP sequence also can be attached to a non-peptide entity such as polymer, fluorophore, solid support, chemical linker, or sugar. In some embodiments, the MAP can be attached to a solid surface or substrate through a linker. In some embodiments, the solid surface or substrate can be polymeric, such as a resin bead or membrane. In some

embodiments, the MAP is incorporated in a longer polypeptide which is further attached to a non-peptide entity, such as a polymeric solid surface. The attachment can be covalent, and can be affected through a linker. Additionally, more than one MAP sequence can be present on a particular molecule or macromolecule. In some embodiments, a lysine-derived linker is attached directly or indirectly to the MAP sequence.

[0056] In some embodiments, the MAP sequence is flanked by additional amino acids. For example, in addition to the MAP tag being an isolated tripeptide XC₁C₂, the MAP tag can comprise a sequence selected from the group consisting of: Zi-XCiC₂; XCiC₂-Z₂; and Z XCiC₂-Z₂, wherin X is any amino acid or amino acid analogue; Q and C₂ are the same or different and are a cysteine, or a cysteine-like non-natural amino acid, or a cysteine-like amino acid analogue; Zi is any amino acid or any sequence of amino acids, and Z₂ is any amino acid or sequence of amino acids that is equivalent or not equivalent to Zi. Non-natural and amino acids analogues can be included as Zi and Z₂. In some embodiments, Zi and Z₂ are both natural amino acids or sequences of natural amino acids. In some embodiments, the MAP sequence is flanked by a lysine. For example, the MAP tag can comprise a sequence selected from the group consisting of K-XC₁C₂ and XCiC₂-K. In some embodiments, the MAP tag can comprise a sequence selected from the group consisting of KNCC and NCCK. In some embodiments, the MAP tag is included within a polypeptide that further comprises a lysine moiety, where the lysine moiety is further linked to a resin through the lysine side chain. Table 1 summarizes illustrative peptides of the invention.

[0057] In some embodiments, a MAP tag of the present disclosure can be encoded in line with a gene or nucleotide sequence that provides for targeted delivery of the MAP tag, either before MAP tag complexation with a metal or after complexation with a metal. Targeted delivery can be accomplished using genes, peptides, or other motifs known to be useful for targeting.

[0058] In some embodiments, MAP tags can be incorporated onto solid surfaces or substrates via natural or synthetic linkers. Additionally, MAPs can be incorporated into a peptide or protein using any synthetic or biosynthetic method for peptide or protein production, wherein the polypeptide or protein is further linked to a polymer or a solid surface or substrate. In some embodiments, one or more MAPs are covalently linked to a synthetic polymer via a polyether linker, such as polyethylene glycol or polypropylene glycol.

[0059] In some embodiments, the MAP tag spontaneously reacts with a metal to form a peptide-metal complex, such as zinc during protein production to increase yield of the desired product. Metal-MAP complexes can form in solution or via transmetallation or any other process.

[0060] In some embodiments, compositions comprise an affinity tag in addition to MAP sequence. The affinity tag can be any affinity tag capable of binding to a substrate. The affinity tag can bind the substrate reversibly or substantially irreversibly. Non-limiting examples of suitable affinity tags include maltose binding protein (MBP), glutathione- S- transferase (GST), poly(His), biotin ligase tags, Strep, HaloTag, cellulose binding domain (CBD), glutathione transferases (GST-tag), and a glycan.

[0061] In some embodiments, the MAP is incorporated in a polypeptide, which further comprises an affinity tag. The present disclosure also provides compositions and methods that provide a substrate for the affinity tag. In such embodiments, the affinity tag and any contaminants that can be bound or retained by the substrate for the affinity tag are separated from the metal product of interest.

[0062] A MAP can be incorporated into a molecule, such as a biomolecule. Non-limiting examples of biomolecules include peptides, proteins, enzymes, growth factors, and antibodies. The MAP can be incorporated before or after binding a metal. The MAP can be incorporated one amino acid at a time, or the entire MAP can be incorporated in a single operation. The MAP can be attached to a wild type biomolecule, inserted into the wild type biomolecule such as by the insertion of amino acids into an amino acid sequence, or the MAP can substitute for a series of amino acids in the wild type biomolecule. A MAP can be attached to a therapeutic molecule, such as any of the foregoing biomolecules, a drug, or drug candidate.

[0063] A MAP can be incorporated into a biomolecule by various techniques. A MAP can be incorporated by a chemical transformation, such as the formation of a covalent bond, such as an amide bond or a peptide bond. A MAP can be incorporated, for example, by solid phase or solution phase peptide synthesis. A MAP can be incorporated by preparing a nucleic acid sequence encoding the biomolecule, wherein the nucleic acid sequence includes a subsequence that encodes the MAP. The subsequence can be in addition to the sequence that encodes the biomolecule, or can substitute for a subsequence of the sequence that encodes the biomolecule.

[0064] A MAP can also be incorporated into a molecule having therapeutic or diagnostic use or potential, by the methods described above. In some embodiments, a therapeutic or diagnostic molecule is a biomolecule. A therapeutic or diagnostic molecule can be natural, synthetic, or semi-synthetic.

Metals.

[0065] A metal can be in elemental form, a metal atom, or a metal ion. Non-limiting examples of metals include transition metals, main group metals, and metals of Group 3, Group 4, Group 5, Group 6, Group 7, Group 8, Group 9, Group 10, Group 11, Group 12, Group 13, Group 14, and Group 15 of the Periodic Table. Non-limiting examples of metal include scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, tin, lead, and bismuth.

Linkers.

[0066] A tag of the invention, such as either a MAP tag or a small organic group, can be attached to a larger molecule, such as a peptide, a protein, or an antibody, through a linker, or directly, in the absence of a linker.

[0067] Direct attachment is possible by covalent attachment of a tag to a region of the larger molecule. For example, the tag could be attached to a terminus of the amino acid sequence of the larger molecule, or could be attached to a side chain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, or glutamic acid residue. The attachment can be via an amide bond, an ester bond, an ether bond, or a thioether bond.

[0068] Attachment via a linker involves incorporation of a linker moiety between the larger molecule and the tag. The tag and the larger molecule can both be covalently attached to the linker. The linker can be cleavable, non-cleavable, self-immolating, hydrophilic, or hydrophobic. The linker has at least two functional groups, one bonded to the larger molecule, and one bonded to the tag, and a linking portion between the two functional groups.

[0069] Non-limiting examples of the functional groups for attachment include functional groups capable of forming, for example, an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond, or a thioether bond. Non-limiting examples of functional groups capable of forming such bonds include amino groups; carboxyl groups; acid halides such as acid fluorides, chlorides, bromides, and iodides; acid anhydrides, including symmetrical, mixed, and cyclic anhydrides; carbonates; carbonyl functionalities bonded to leaving groups such as cyano, succinimidyl, and iV-hydroxysuccinimidyl; hydroxyl groups; sulfhydryl groups; and molecules possessing, for example, alkyl, alkenyl, alkynyl, allylic, or benzylic leaving groups, such as halides, mesylates, tosylates, triflates, epoxides, phosphate esters, sulfate esters, and besylates.

[0070] Non-limiting examples of the linking portion include alkylene, alkenylene, alkynylene, polyether, such as polyethylene glycol (PEG), polyester, polyamide, and polyamine, any of which being unsubstituted or substituted with any number of substituents, such as halogens, hydroxyl groups, sulfhydryl groups, amino groups, nitro groups, nitroso groups, cyano groups, azido groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenyl groups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, urethane groups, epoxides, and ester groups.

0071] Non-limiting examples of linkers include:

, wherein each n is independently 0 to about

I, 000; 1 to about 1,000; 0 to about 500; 1 to about 500; 0 to about 250; 1 to about 250; 0 to about 200; 1 to about 200; 0 to about 150; 1 to about 150; 0 to about 100; 1 to about 100; 0 to about 50; 1 to about 50; 0 to about 40; 1 to about 40; 0 to about 30; 1 to about 30; 0 to about 25; 1 to about 25; 0 to about 20; 1 to about 20; 0 to about 15; 1 to about 15; 0 to about 10; 1 to about 10; 0 to about 5; or 1 to about 5. In some embodiments, each n is independently 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about

I I, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. In some embodiments, m is 1 to about 1,000; 1 to about 500; 1 to about 250; 1 to about 200; 1 to about 150; 1 to about 100; 1 to about 50; 1 to about 40; 1 to about 30; 1 to about 25; 1 to about 20; 1 to about 15; 1 to about 10; or 1 to about 5. In some embodiments, m is 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50.

Homology.

[0072] A peptide of the invention can be prepared, for example, by peptide synthesis or expression of an appropriate nucleic acid molecule. Non limiting examples of peptide sequencing methods include: a) liquid-phase peptide synthesis; b) solid-phase peptide synthesis, with a polystyrene resin, a polyamide resin, a PEG hybrid polystyrene resin, a PEG base resin, and/or a combination of any solid phase support; and c) synthetic biology. Non- limiting examples of methods for the expression of an appropriate nucleic acid molecule include molecular cloning and recombinant DNA technologies.

[0073] A peptide of the invention prepared by peptide synthesis can have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, or at least 30% homology with a naturally occurring peptide or a parent peptide. A peptide of the invention prepared by the expression of an appropriate nucleic acid molecule can have at least 99.99%, at least 99.9%, at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, or at least 30% homology with a naturally occurring peptide or a parent peptide.

[0074] A nucleic acid sequence encoding a peptide of the invention can have at least 99.99%, at least 99.9%, at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, or at least 30% homology with a naturally occurring nucleic acid sequence or a parent nucleic acid sequence. An appropriate nucleic acid sequence for the preparation of a peptide of the invention can be a degenerate sequence. The percent homology between sequences can be calculated using a plurality of algorithms.

[0075] A peptide of the invention can be covalently incorporated into a known molecule without disrupting certain biochemical properties of the molecule. For example, a peptide of the invention can be covalently incorporated into a molecule such that the structure of the original beta-sheet and alpha-helices of the molecule are not disrupted. A peptide of the invention can be covalently incorporated into a molecule such that the original three- dimensional structure of the molecule is preserved. A peptide of the invention can be covalently incorporated into a molecule such that the original arrangement of multi-subunit complexes, or quaternary structure, is preserved. In some embodiments, a peptide of the invention can change the solubility of an existing molecule by providing a method to change the net charge of a molecule without disturbing the original secondary, tertiary or quaternary structure. Therapeutic Uses.

[0076] The incorporation of a metal abstracting peptide with a high affinity for binding metal can significantly improve the efficacy of a potential therapeutic for a cancer mediated by a metalloenzyme, for example, MMPs. A plurality of subjects in need or want of treatment for the cancer can benefit from the use of a greatly improved therapy. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, or infants.

[0077] In some embodiments, a peptide of the invention is used in the treatment of a cancer mediated by a metalloenzyme. Non-limiting examples of cancers mediated by a

metalloenzyme include, ovarian cancer, colorectal cancer, Waldenstrom's macro globulinemia (cancer of B lymphocytes), head and neck cancer, lung cancer, hepatocellular carcinoma, breast cancer, superficial bladder cancer, gastric cancer, renal cancer, pancreatic cancer, neuroectodermal cancer, cervical cancer, prostate cancer, melanoma, and sarcoma.

[0078] In some embodiments, a composition of the invention is used in the treatment of a solid tumor associated with a metalloenzyme. In some embodiments, a composition of the invention is used to prevent growth of a solid tumor associated with a metalloenzyme. In some embodiments, a composition of the invention is used to prevent a metastasis of a cancer associated with a metalloenzyme.

[0079] In some embodiments, the invention provides a use of a peptide or a

pharmaceutically-acceptable salt thereof in the preparation of a medicament for inhibiting a metalloenzyme, wherein the peptide or the pharmaceutically-acceptable salt thereof binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[0080] In some embodiments, the invention provides a use of a peptide or a

pharmaceutically-acceptable salt thereof for inhibiting a metalloenzyme in a subject in need thereof, wherein the peptide or the pharmaceutically-acceptable salt thereof binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[0081] In some embodiments, the invention provides a use of a peptide or a

pharmaceutically-acceptable salt thereof in the preparation of a medicament for the treatment of a cancer associated with a metalloenzyme, wherein the peptide or the pharmaceutically- acceptable salt thereof binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[0082] In some embodiments, the invention provides a use of a peptide or a

pharmaceutically-acceptable salt thereof in the treatment of a cancer associated with a metalloenzyme in a subject in need thereof, wherein the peptide or the pharmaceutically- acceptable salt thereof binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

Pharmaceutical Formulations.

[0083] A pharmaceutical composition of the disclosure can be a combination of any pharmaceutical compounds described herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions can be administered in therapeutically-effective amounts as pharmaceutical compositions by any form and route known in the art including, for example, intravenous, subcutaneous, intramuscular, oral, rectal, parenteral, ophthalmic, pulmonary, transdermal, vaginal, otic, nasal, and topical administration.

[0084] A pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the compound directly into an organ, optionally in a depot or sustained release formulation. Pharmaceutical compositions can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. A rapid release form can provide an immediate release. An extended release formulation can provide a controlled release or a sustained delayed release.

[0085] For oral administration, pharmaceutical compositions can be formulated by combining the active compounds with pharmaceutically acceptable carriers or excipients. Such carriers can be used to formulate liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a subject. Non-limiting examples of solvents used in an oral dissolvable formulation can include water, ethanol, isopropanol, saline, physiological saline, DMSO, dimethylformamide, potassium phosphate buffer, phosphate buffer saline (PBS), sodium phosphate buffer, 4-2-hydroxyethyl-l-piperazineethanesulfonic acid buffer (HEPES), 3-(N-morpholino)propanesulfonic acid buffer (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) buffer (PIPES), and saline sodium citrate buffer (SSC). Non-limiting examples of co- solvents used in an oral dissolvable formulation can include sucrose, urea, cremaphor, DMSO, and potassium phosphate buffer.

[0086] Pharmaceutical preparations can be formulated for intravenous administration. The pharmaceutical compositions can be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0087] The active compounds can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams, and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0088] The compounds can also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as a mixture of fatty acid glycerides, optionally in combination with cocoa butter, is first melted.

[0089] In practicing the methods of treatment or use provided herein, therapeutically- effective amounts of the compounds described herein are administered in pharmaceutical compositions to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal such as a human. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

[0090] Pharmaceutical compositions can be formulated using one or more physiologically- acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Formulation can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising compounds described herein can be manufactured in a conventional manner, for example, by means of conventional mixing, dissolving, granulating, or emulsifying.

[0091] The pharmaceutical compositions can include at least one pharmaceutically acceptable carrier, diluent, or excipient and compounds described herein or pharmaceutically- acceptable salt form.

[0092] Methods for the preparation of compositions comprising the compounds described herein can include formulating the compounds with one or more inert, pharmaceutically- acceptable excipients. Liquid compositions include, for example, solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein. Semi-solid compositions include, for example, gels, suspensions and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically- acceptable additives.

[0093] Compounds can be delivered via liposomal technology. The use of liposomes as drug carriers can increase the therapeutic index of the compounds. Liposomes are composed of natural phospholipids, and can contain mixed lipid chains with surfactant properties, for example, egg phosphatidylethanolamine. A liposome design can employ surface ligands for attaching to unhealthy tissue. Non-limiting examples of liposomes include the multilamellar vesicle (MLV), the small unilamellar vesicle (SUV), and the large unilamellar vesicle (LUV). Liposomal physicochemical properties can be modulated to optimize penetration through biological barriers and retention at the site of administration, and to prevent premature degradation and toxicity to non-target tissues. Optimal liposomal properties depend on the administration route: large-sized liposomes show good retention upon local injection, small- sized liposomes are better suited to achieve passive targeting. PEGylation reduces the uptake of the liposomes by liver and spleen, and increases the circulation time, resulting in increased localization at the inflamed site due to the enhanced permeability and retention (EPR) effect. Additionally, liposomal surfaces can be modified to achieve selective delivery of the encapsulated drug to specific target cells. Non-limiting examples of targeting ligands include monoclonal antibodies, vitamins, peptides, and polysaccharides specific for receptors concentrated on the surface of cells associated with the disease.

[0094] Non-limiting examples of dosage forms suitable for use in the disclosure include liquid, elixir, nanosuspension, aqueous or oily suspensions, drops, syrups, and any

combination thereof. Non-limiting examples of pharmaceutically-acceptable excipients suitable for use in the disclosure include granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, glidants, anti- adherents, anti-static agents, surfactants, anti- oxidants, gums, coating agents, coloring agents, flavouring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, plant cellulosic material and spheronization agents, and any combination thereof.

[0095] Non-limiting examples of pharmaceutically-acceptable excipients can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &

Wilkinsl999), each of which is incorporated by reference in its entirety.

[0096] Compositions of the invention can be packaged as a kit. In some embodiments, a kit includes written instructions on the administration/use of the composition. The written material can be, for example, a label. The written material can suggest conditions methods of administration. The instructions provide the subject and the supervising physician with the best guidance for achieving the optimal clinical outcome from the administration of the therapy. The written material can be a label. In some embodiments, the label can be approved by a regulatory agency, for example the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or other regulatory agencies.

[0097] In some embodiments, a kit of the invention comprises: a) a peptide comprising a sequence XC₁C₂, wherein X is any natural or non-natural amino acid or amino acid analogue, and Ci and C₂ are each individually chosen from a cysteine and a sulfur-containing alpha or beta amino acid, wherein a molecule is bound to the peptide; b) a metal; and c) written instructions describing a use of the kit in treatment of a condition.

Dosing.

[0098] Pharmaceutical compositions described herein can be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compounds. The unit dosage can be in the form of a package containing discrete quantities of the formulation. Non- limiting examples are liquids in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Multiple-dose reclosable containers can be used, for example, in combination with a preservative. Formulations for parenteral injection can be presented in unit dosage form, for example, in ampoules, or in multi-dose containers with a preservative.

[0099] A compound described herein can be present in a composition in a range of from about 1 mg to about 2000 mg; from about 100 mg to about 2000 mg; from about 10 mg to about 2000 mg; from about 5 mg to about 1000 mg, from about 10 mg to about 500 mg, from about 50 mg to about 250 mg, from about 100 mg to about 200 mg, from about 1 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 150 mg, from about 150 mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mg to about 300 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg, from about 400 mg to about 450 mg, from about 450 mg to about 500 mg, from about 500 mg to about 550 mg, from about 550 mg to about 600 mg, from about 600 mg to about 650 mg, from about 650 mg to about 700 mg, from about 700 mg to about 750 mg, from about 750 mg to about 800 mg, from about 800 mg to about 850 mg, from about 850 mg to about 900 mg, from about 900 mg to about 950 mg, or from about 950 mg to about 1000 mg.

[00100] A compound described herein can be present in a composition in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg, about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450 mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg.

[00101] In some embodiments, a dose can be expressed in terms of an amount of the drug divided by the mass of the subject, for example, miligrams of drug per kilograms of subject body mass. In some embodiments, a platinum containing compound attached to a metal abstracting peptide (MAP) is present in a composition in an amount ranging from about 250 mg/kg to about 2000 mg/kg, about 10 mg/kg to about 800 mg/kg, about 50 mg/kg to about 400 mg/kg, about 100 mg/kg to about 300 mg/kg, or about 150 mg/kg to about 200 mg/kg.

[00102] The foregoing ranges are merely suggestive. Dosages can be altered depending on a number of variables, including, for example, the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

Pharmaceutically-Acceptable Salts.

[00103] The invention provides the use of pharmaceutically-acceptable salts of any therapeutic compound described herein. Pharmaceutically-acceptable salts include, for example, acid- addition salts and base-addition salts. The acid that is added to the compound to form an acid-addition salt can be an organic acid or an inorganic acid. A base that is added to the compound to form a base-addition salt can be an organic base or an inorganic base. In some embodiments, a pharmaceutically-acceptable salt is a metal salt. In some embodiments, a pharmaceutically-acceptable salt is an ammonium salt.

[00104] Metal salts can arise from the addition of an inorganic base to a compound of the invention. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. In some

embodiments, the metal is lithium, sodium, potassium, cesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminum, copper, cadmium, or zinc.

[00105] In some embodiments, a metal salt is a lithium salt, a sodium salt, a potassium salt, a cesium salt, a cerium salt, a magnesium salt, a manganese salt, a iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, an aluminum salt, a copper salt, a cadmium salt, or a zinc salt.

[00106] Ammonium salts can arise from the addition of ammonia or an organic amine to a compound of the invention. In some embodiments, the organic amine is triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N- methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine, piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, or pipyrazine.

[00107] In some embodiments, an ammonium salt is a triethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N-methylpiperidine salt, an N- ethylpiperidine salt, a dibenzylamine salt, a piperazine salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, or a pipyrazine salt.

[00108] Acid addition salts can arise from the addition of an acid to a compound of the invention. In some embodiments, the acid is organic. In some embodiments, the acid is inorganic. In some embodiments, the acid is hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, nitrous acid, sulfuric acid, sulfurous acid, a phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, gentisinic acid, gluconic acid, glucaronic acid, saccaric acid, formic acid, benzoic acid, glutamic acid, pantothenic acid, acetic acid, propionic acid, butyric acid, fumaric acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, oxalic acid, or maleic acid.

[00109] In some embodiments, the salt is a hydrochloride salt, a hydrobromide salt, a hydroiodide salt, a nitrate salt, a nitrite salt, a sulfate salt, a sulfite salt, a phosphate salt, isonicotinate salt, a lactate salt, a salicylate salt, a tartrate salt, an ascorbate salt, a gentisinate salt, a gluconate salt, a glucaronate salt, a saccarate salt, a formate salt, a benzoate salt, a glutamate salt, a pantothenate salt, an acetate salt, a propionate salt, a butyrate salt, a fumarate salt, a succinate salt, a methanesulfonate (mesylate) salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt, a citrate salt, an oxalate salt , or a maleate salt.

EXAMPLES

Example 1: Peptides used to inhibit MMP-8.

[00110] To ensure accessibility of the inhibitory peptide module and permit interaction with MMP-8 once tethered to the polymer, the MAP peptide NCC was incorporated into a longer polypeptide to provide a spacer between the inhibitory MAP module and the polymer surface. A thirteen amino acid spacer was chosen that included a tryptophan for

quantification. A sixteen amino acid long peptide called tether- MAP (Ser-Trp-Leu- Ala-Tyr- Pro-Gly-Ala-Val-Ser-Tyr-Arg-Gly-Asn-Cys-Cys) (SEQ ID No. 1) was generated with MAP peptide Asn-Cys-Cys at the C-terminus.

[00111] Peptides used to inhibit MMP-8 in this study include the MAP peptide NCC and tether-MAP (SEQ ID No. 1).

Example 2: Preparation of polymer resins.

[00112] Polymer resins with peptide grafting capability were prepared using standard adhesive formulation of 2-hydroxyethyl methacrylate (HEMA) and 2,2-bis[4-(2-hydroxy-3- methacryloxypropoxy)phenyl] -propane (Bis-GMA) doped with different wt % of 2- Aminoethyl methacrylate hydrochloride (AEMA) monomer.

[00113] All the materials were used as received. The photoinitiators used in this study were 0.5 % w/w each camphorquinone (CQ), ethyl 4-(dimethylamino)benzoate (EDMAB) and diphenyliodonium hexafluorophosphate (DPIHP). [00114] The control/standard adhesive consisted of HEMA and BisGMA with a mass ratio of 60/40 % w/w (HEMA/ BisGMA). The experimental adhesives were formulated with 5, 10, 15 and 20 % w/w AEMA monomer to facilitate grafting onto the poymer surface.

[00115] Mixtures of monomers/photoinitiators were prepared in a brown glass vial in the absence of visible light. The solutions containing the monomers/photoinitiators were mixed overnight at room temperature to promote complete dissolution and formation of a homogeneous solution prior to light-induced polymerization.

Example 3: Degree of conversion of polymer resins.

[00116] To determine the effect of altering the standard formulation to incorporate

AEMA, the degree of conversion (DC) for each resin formulation was examined.

[00117] The DC and polymerization behavior was determined by Fourier Transform

Infrared Spectrometry (FTIR). Real-time in situ monitoring of the photopolymerization of the different adhesive solutions was performed using an infrared spectrometer (Spectrum 400 Fourier transform infrared spectrophotometer, Perkin-ElmerTM, Waltham, MA) at a resolution of 4 cm^"1. One drop of adhesive solution was placed on the diamond crystal top plate of an attenuated total reflectance (ATR) accessory (PIKE Technologies Gladi-ATR, Madison, WI) and covered with a cover slip to prevent oxygen inhibition of polymerization. A 40-s exposure to the commercial visible-light-polymerization unit (Spectrum VR 800, Dentsply, Milford, DE) at an intensity of 550 mW cm^" was initiated after 50 spectra had been recorded. Real-time IR spectra were continuously recorded for 900 s after light activation began. A time-based spectrum collector (Spectrum TimeBase, Perkin-ElmerTM) was used for continuous and automatic collection of spectra during polymerization. Three replicates were obtained for each adhesive formulation. The change of the band ratio profile [1637 cm^"1 (C=C)/1608 cm^"1 (phenyl)] was monitored and degree of conversion (DC) was calculated using the following equation, where A= absorbance, based on the decrease in the absorption intensity band ratio before and after light curing.

DC =

[00118] AEMA is poorly soluble in BisGMA, thus the control resin had a higher proportion of HEMA than is standard in order to accommodate up to 20% AEMA. Real-time photo-polymerization kinetic behavior of the control and experimental adhesives was analyzed. With increasing AEMA content, DC increased from 61% for the control resin to 70-80% for the experimental resins. At each level of AEMA content, the polymerization rates of the experimental adhesives were significantly higher than those of the control. The standard adhesive formulation, however, had a DC of about 70%, which was well matched by the 15-20% AEMA-containing samples.

Example 4: Resin coating of 96-well plate.

[00119] The adhesive resin formulations were polymerized in 96-well plates in order to carry out grafting and inhibition studies.

[00120] To make polymer coating on a 96-well plate, 40 μΐ_^ of each resin formulation was aliquoted in different wells and cured with a 40-s exposure to the commercial visible- light-polymerization unit (Spectrum VR 800, Dentsply™, Milford, DE) at an intensity of 550 mW cm^"2. The plate was left under dark for post polymerization for 48 hours.

[00121] Polymerization and curing in the 96-well plates were performed using atmospheric conditions and oxygen-depleted conditions in which the plate was placed in a sealed enclosure and purged with nitrogen for 5 min. Samples produced with and without oxygen depletion behaved comparably.

Example 5: Methyl orange assay.

[00122] A 0.2% methyl orange (Fisher Scientific™) stock solution was prepared using deionized water. This solution was diluted to 0.05% with 0.1 M NaH₂P0₄ solution (solid from Fisher Scientific) to prepare an acidic methyl orange solution (final buffer concentration was 0.075M NaH₂P0₄ solution).

[00123] Polymers were presoaked in water for 2-7 days to remove any leachables.

Polymers were removed from water, rinsed under a stream of deionized water for

approximately 10-30 seconds, and excess water was removed by blotting samples. Polymers were then incubated in acidic methyl orange solution for approximately one hour. Polymers were removed from solution, rinsed under a stream of deionized water for approximately 10- 30 seconds, and excess water was removed by blotting samples. A 0.1 M Na₂C0₃ (Fisher Scientific™) solution was prepared (base solution). Polymers were incubated in 5 milliliters of base solution for approximately 6.5 hours. The absorbance of the resultant base solution (with methyl orange extracted from the polymer samples) was measured using a UV-Vis spectrophotometer (Thermo Scientific Evolution300 UV-VIS) from 400-800 nm. Methyl orange has a maximum absorbance at 465 nm. The concentration was determined by using a standard calibration curve with the dye solution at different concentrations. This curve was linear over the concentration range investigated here. The concentration can be used to determine the amount of dye in the solution, which can be used to estimate the number of adsorbed methyl orange molecules per mass of polymer.

Example 6: Determination of solvent-accessible amine content.

[00124] Methyl orange assay (as described in Example 5) was used to determine the solvent accessible amine content in the polymers.

[00125] Methyl orange electrostatically bound to the available amines within the polymer and was extracted using basic solution. UV-Vis analysis of the series of extracts containing methyl orange had increased absorbance at 465 nm, and the intensities reflected the increasing amount of solvent accessible amines within the polymer, corresponding to the expected percent increase in incorporated amine within the polymer. FIGURE 3 panel a depicts that correlation, indicating the increase in amine content resulted in a proportional increase in methyl orange adsorption and extraction from the polymers. This observation confirmed that a higher percentage of amine within the polymer formulation resulted in additional solution-accessible amine sites incorporated within the polymer.

Example 7: Peptide grafting on polymer surfaces.

[00126] Disuccinimidyl suberate (DSS) linker chemistry was used to graft the inhibitor peptide onto the polymer surfaces via amine-containing residues. DSS is a homobifunctional linker, thus self-coupling between soluble peptides occurs readily in solution. A series of experimental conditions were examined to optimize the grafting reaction.

[00127] 47 μΐ. of HEPES buffer pH 7.4, 2 of 1 mM peptide and 1 of 50 mM

DSS linker were used per well. The plate was incubated at room temp for 1 h and the reaction was quenched by adding 50 μΐ_^ of 20 mM Tris buffer for 15 minutes. Wells were washed ten times with 400 μΐ_^ dd H₂0 prior to data acquisition. A positive control was performed with 4'-(aminomethyl) fluorescein hydrochloride (Invitrogen) under same conditions over all polymer surfaces.

[00128] Grafting of tether-MAP (SEQ ID No. 1), monitored by intrinsic tryptophan fluorescence (E_ex/Eem = 280/360 nm), followed an almost linear trend of RFU with AEMA content in polymer resin as illustrated in FIGURE 3 panel b. The positive control with FITC, measured at E_ex/E_em = 492/516, followed the same trend as shown in FIGURE 3 panel b. The findings indicated that 10 mM DSS and 0.1 mM peptide/dye are the optimum condition for efficient grafting. These results paralleled the trend observed for solvent accessible amine content, determined using the methyl orange assay

Example 8: MMP-8 activity RED fluorometric assay.

[00129] MMP-8 Fluorometric Drug Discovery Kit, RED was used to study MMP-8 inhibition in solution and on polymer surfaces. This kit also contained the same standard inhibitor NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid) as in the colorimetric kit. The components were diluted in assay buffer warmed to reaction

temperature 37 °C similarly as for colorimetric kit. The fluorescent microplate reader was calibrated using Ex/Em = 545/576 nm, with cutoff set at 570 nm.

[00130] The assays were performed in a convenient 96-well microplate format. NNGH was diluted 1/200 in assay buffer to required total volume and warmed to reaction

temperature 37 °C. Substrate peptide was diluted 1/50 in assay buffer to required total volume. MMP-8 enzyme was diluted 1/100 in assay buffer to required total volume and warmed to reaction temperature 37 °C. Assay buffer was pipetted into each desired well as follows: Blank (no MMP-8) = 90 μΐ_^ assay buffer, control (no inhibitor) = 70 μΐ_^ assay buffer and inhibitor NNGH and test inhibitors = 50 μΐ_^ assay buffer. 20 μΐ_^ of MMP-8 was added in all wells except the blank, followed by 20 μΐ_^ of inhibitor NNGH (final inhibitor

concentration^.3 μΜ) in NNGH designated wells and different concentrations of test inhibitor peptides (final concentrations of 0.01, 0.1, 1.0 μΜ) in respective wells. The plate was incubated for 90 minutes at reaction temperature 37 °C to allow inhibitor/enzyme interaction and then 10 μΐ_^ of substrate peptide (diluted and equilibrated to reaction temperature) was added (final substrate concentration^ 00 μΜ) to start the reaction. The plate was read continuously in the fluorescent microplate reader, using Ex/Em = 545/576 nm, with cutoff set at 570 nm at set reaction temperature 37 °C.

Example 9: MMP-8 inhibitor analysis in solution.

[00131] To examine the ability of the peptides NCC and tether-MAP (SEQ ID No. 1) to inhibit MMP-8, the RED fluorimetric (described in Example 8) assay was carried out in solution. The peptides were tested and compared with positive and negative control reactions.

[00132] As illustrated in FIGURE 2 panel a, 0.10 μΜ of MAP peptide NCC achieved nearly complete inhibition of MMP-8 in solution. 1.3 μΜ of the standard inhibitor NNGH was required to achieve a similar result, whereas 0.1 μΜ NNGH achieved about 50% inhibition in similar conditions. This result suggests that MAP peptide NCC is an effective inhibitor of MMP-8.

[00133] The tether-MAP (SEQ ID No. 1) peptide was tested for the ability to inhibit

MMP-8 activity in solution. tether-MAP (SEQ ID No. 1) was found to require aboutlO-fold higher concentration than the MAP peptide NCC alone. As illustrated in FIGURE 2 panel b, the tether-MAP (SEQ ID No. 1) peptide completely abrogated turn over at a comparable or slightly-lower concentration than did NNGH.

Example 10: MMP-8 inhibitor analysis on polymer surface.

[00134] To verify that the peptide remains functional as an inhibitor after tethering to the polymer surface, the MMP-8 assay was carried out on the series of peptide- grafted polymer surfaces, including control resin, 5%, 10%, and 20% AEMA-doped resin. Each resin was first modified with tether-MAP (SEQ ID No. 1) peptide and then assayed as in solution and compared with control and NNGH reactions. In addition, the control and standard inhibitor reactions were run on the series of unconjugated, bare polymer resins to account for background and establish any differences caused by changes in polymer composition. All resins behaved comparably, indicating the polymer does not affect the assay and that the observed inhibition results from the grafted peptide. FIGURE 4 illustrates that the efficiency of inhibition increased with increasing amount of grafted peptide, corresponding to the percent amine in the formulation. The 20% AEMA containing resin grafted with tether-MAP (SEQ ID No. 1) showed the best result, in which complete inhibition of MMP-8 was achieved.

EMBODIMENTS

[00135] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[00136] Embodiment 1. A method of inhibiting a metalloenzyme in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a composition comprising a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[00137] Embodiment 2. The method of Embodiment 1, wherein the metalloenzyme is a matrix metalloproteinase. [00138] Embodiment 3. The method of Embodiment 2, wherein the matrix metalloproteinase is a matrix metalloproteinase-8 (MMP-8).

[00139] Embodiment 4. The method of any one of Embodiments 1-3, wherein the metal is zinc.

[00140] Embodiment 5. The method of any one of Embodiments 1-4, wherein one of the pair of neighboring sulfur-containing amino acid residues is a cysteine residue.

[00141] Embodiment 6. The method of any one of Embodiments 1-4, wherein both of the pair of neighboring sulfur-containing amino acid residues are cysteine residues.

[00142] Embodiment 7. The method of any one of Embodiments 1-6, wherein the peptide or the pharmaceutically-acceptable salt thereof is administered in a unit dosage form, wherein the unit dosage form further comprises a pharmaceutically-acceptable excipient.

[00143] Embodiment 8. The method of any one of Embodiments 1-7, wherein the therapeutically-effective amount is from about 50 mg to about 100 mg.

[00144] Embodiment 9. The method of any one of Embodiments 1-8, wherein the administration is intravenous.

[00145] Embodiment 10. The method of any one of Embodiments 1-8, wherein the administration is oral.

[00146] Embodiment 11. The method of any one of Embodiments 1-10, wherein the subject is a human.

[00147] Embodiment 12. A method of treating a cancer associated with a

metalloenzyme in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a composition comprising a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

[00148] Embodiment 13. The method of Embodiment 12, wherein the metalloenzyme is a matrix metalloproteinase.

[00149] Embodiment 14. The method of Embodiment 13, wherein the matrix metalloproteinase is a matrix metalloproteinase-8 (MMP-8).

[00150] Embodiment 15. The method of any one of Embodiments 12-14, wherein the metal is zinc.

[00151] Embodiment 16. The method of any one of Embodiments 12-15, wherein one of the pair of neighboring sulfur-containing amino acid residues is a cysteine residue.

[00152] Embodiment 17. The method of any one of Embodiments 12-15, wherein both of the pair of neighboring sulfur-containing amino acid residues are cysteine residues. [00153] Embodiment 18. The method of any one of Embodiments 12-17, wherein the peptide or the pharmaceutically-acceptable salt thereof is administered in a unit dosage form, wherein the unit dosage form further comprises a pharmaceutically-acceptable excipient.

[00154] Embodiment 19. The method of any one of Embodiments 12-18, wherein the cancer is ovarian cancer.

[00155] Embodiment 20. The method of any one of Embodiments 12-18, wherein the cancer is colorectal cancer.

[00156] Embodiment 21. The method of any one of Embodiments 12-18, wherein the cancer is lung cancer.

[00157] Embodiment 22. The method of any one of Embodiments 12-18, wherein the cancer is breast cancer.

[00158] Embodiment 23. The method of any one of Embodiments 12-18, wherein the cancer is a melanoma.

[00159] Embodiment 24. The method of any one of Embodiments 12-23, wherein the administration is intravenous.

[00160] Embodiment 25. The method of any one of Embodiments 12-23, wherein the administration is oral.

[00161] Embodiment 26. The method of any one of Embodiments 12-25, wherein the subject is a human.

[00162] Embodiment 27. A method of inhibiting a metalloenzyme, the method comprising contacting the metalloenzyme with a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur- containing amino acid residues in the peptide.

[00163] Embodiment 28. The method of Embodiment 27, wherein the metalloenzyme is a matrix metalloproteinase.

[00164] Embodiment 29. The method of Embodiment 28, wherein the matrix metalloproteinase is a matrix metalloproteinase-8 (MMP-8).

[00165] Embodiment 30. The method of any one of Embodiments 27-29, wherein the metal is zinc.

[00166] Embodiment 31. The method of any one of Embodiments 27-30, wherein one of the pair of neighboring sulfur-containing amino acid residues is a cysteine residue.

[00167] Embodiment 32. The method of any one of Embodiments 27-30, wherein both of the pair of neighboring sulfur-containing amino acid residues are cysteine residues. [00168] Embodiment 33. The method of any one of Embodiments 27-32, wherein the inhibiting comprises reducing enzymatic activity of the metalloenzyme.

[00169] Embodiment 34. The method of Embodiment 33, wherein the reducing comprises reducing enzymatic activity of the metalloenzyme by at least 30% compared to a wild- type enzyme.

[00170] Embodiment 35. The method of Embodiment 33, wherein the reducing comprises reducing enzymatic activity of the metalloenzyme by at least 70% compared to a wild- type enzyme.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of inhibiting a metalloenzyme in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a composition comprising a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

2. The method of claim 1, wherein the metalloenzyme is a matrix metalloproteinase.

3. The method of claim 2, wherein the matrix metalloproteinase is a matrix

metalloproteinase-8 (MMP-8).

4. The method of claim 1, wherein the metal is zinc.

5. The method of claim 1, wherein one of the pair of neighboring sulfur-containing amino acid residues is a cysteine residue.

6. The method of claim 1, wherein both of the pair of neighboring sulfur-containing amino acid residues are cysteine residues.

7. The method of claim 1, wherein the peptide or the pharmaceutically-acceptable salt thereof is administered in a unit dosage form, wherein the unit dosage form further comprises a pharmaceutically-acceptable excipient.

8. The method of claim 1, wherein the therapeutically-effective amount is from about 50 mg to about 100 mg.

9. The method of claim 1, wherein the administration is intravenous.

10. The method of claim 1, wherein the administration is oral.

11. The method of claim 1, wherein the subject is a human.

12. A method of treating a cancer associated with a metalloenzyme in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of a composition comprising a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

13. The method of claim 12, wherein the metalloenzyme is a matrix metalloproteinase.

14. The method of claim 13, wherein the matrix metalloproteinase is a matrix

metalloproteinase-8 (MMP-8).

15. The method of claim 12, wherein the metal is zinc.

16. The method of claim 12, wherein one of the pair of neighboring sulfur-containing amino acid residues is a cysteine residue.

17. The method of claim 12, wherein both of the pair of neighboring sulfur-containing amino acid residues are cysteine residues.

18. The method of claim 12, wherein the peptide or the pharmaceutically-acceptable salt thereof is administered in a unit dosage form, wherein the unit dosage form further comprises a pharmaceutically-acceptable excipient.

19. The method of claim 12, wherein the cancer is ovarian cancer.

20. The method of claim 12, wherein the cancer is colorectal cancer.

21. The method of claim 12, wherein the cancer is lung cancer.

22. The method of claim 12, wherein the cancer is breast cancer.

23. The method of claim 12, wherein the cancer is a melanoma.

24. The method of claim 12, wherein the administration is intravenous.

25. The method of claim 12, wherein the administration is oral.

26. The method of claim 12, wherein the subject is a human.

27. A method of inhibiting a metalloenzyme, the method comprising contacting the metalloenzyme with a peptide or a pharmaceutically-acceptable salt thereof that binds a metal of the metalloenzyme with each of a pair of neighboring sulfur-containing amino acid residues in the peptide.

28. The method of claim 27, wherein the metalloenzyme is a matrix metalloproteinase.

29. The method of claim 28, wherein the matrix metalloproteinase is a matrix

metalloproteinase-8 (MMP-8).

30. The method of claim 27, wherein the metal is zinc.

31. The method of claim 27, wherein one of the pair of neighboring sulfur-containing amino acid residues is a cysteine residue.

32. The method of claim 27, wherein both of the pair of neighboring sulfur-containing amino acid residues are cysteine residues.

33. The method of claim 27, wherein the inhibiting comprises reducing enzymatic activity of the metalloenzyme.

34. The method of claim 33, wherein the reducing comprises reducing enzymatic activity of the metalloenzyme by at least 30% compared to a wild-type enzyme.

35. The method of claim 33, wherein the reducing comprises reducing enzymatic activity of the metalloenzyme by at least 70% compared to a wild-type enzyme.