WO2008017955A1 - Novel phosphopeptides and uses thereof in preventing kidney stone formation - Google Patents

Abstract

Inhibitory phosphopeptide compositions and methods for preventing the formation and growth of calcium oxalate monohydrate and hydroxyapatite containing crystals are provided.

Description

NOVEL PHOSPHOPEPTIDES AND USES THEREOF IN PREVENTING KIDNEY

STONE FORMATION

Related Applications

This application claims priority to U.S. Provisional Application No. 60/774,3Sl, filed on February 17, 2006, the content of which is specifically incorporated herein by reference.

Background of the Invention

Kidney stones represent a widespread, painful medical problem that is believed to have increased over the last few decades, particularly among Caucasian males in the United States and other predominantly Caucasian-populated nations. The most common type of kidney stone, or renal stone, is idiopathic in origin and generally calcareous. In contrast to the normal biomineralization of bones and teeth, the calcium oxalate biomineralization which is believed to cause renal stones contributes significantly to the cost of health care in the United States. Problems associated with renal stones not only include the perception of pain in the afflicted patient, but also mechanical irritation and compromise of renal tissue, back-pressure from restricted urine flow, and risk of infection due to mechanical irritation or back-pressure or to the mere presence of a foreign body in the kidney.

Ironically, it is believed that crystallization within the urinary tract occurs on an ongoing basis, with the formation of small crystals providing the usual excretory function for eliminating calcareous stone salts. It is when such crystallization is not restricted to the urinary tract that the formation of unwanted crystalline stones in the kidney occurs. There are various theories as to why the crystallization is not restricted to the urinary tract in certain individuals, including genetic and dietary causes, but ultimately the cause of renal stones is not fundamentally understood. This idiopathy of renal stones creates unique challenges in developing effective treatments, because a general treatment must be able to inhibit formations potentially attributable to a variety of causes.

Known methods of treating kidney stones include lithotripsy, chemical irrigation for partial or complete dissolution, surgical interventions and other techniques. Lithotripsy alone is performed on about 500,000 residents of the United States every year, and the costs involved in this lithotripsy medical care and concomitant lost productivity are enormous. Certain citric acid and citrate derivative pharmaceutical compositions have been developed to solubilize the salts and dissolve calcareous formations. However, a need remains for a simple, noninvasive, outpatient approach for inhibiting the growth of kidney stones and discouraging recurrence on a maintenance basis

Summary of the Invention In one aspect, the invention features novel phosphopeptides that are capable of inhibiting the formation and growth of calcium oxalate- and/or hydroxyapatite-containing crystals. Certain phosphopeptides are comprised of in the range of 10 - 20 amino acids am consist of the following consensus sequences: NNNNNNNTNNNNNNpNNN (SEQ ID NO: 6); NpNNNNNNONfNNNNNpNNN (SEQ ID NO: 7); NNNN_PNNNNNNNNN_PNNN (SEQ ID NO: 8), and NpNNN_PNNNNNNNNN_PNNN (SEQ ID NO: 9). The phosphorylated residue(s) (-Np-) may include phosphoserine, phosphotyrosine, or phosphothreonine. Th phosphorylated residues may be contiguous and adjacent to acidic amino acids. Other phosphopeptides are comprised of at least about 25% of acidic amino acids (such aspartic acid, glutamic acidor gamma carboxyglutamic acid), no more than about 10% basic amino acids (such as lysine or arginine) and at least about 15% phosphorylated amino acids, such as phosphoserine, phosphothreonine or phosphotyrosine. Still other phosphopeptides are phosphorylated forms of SHESTEQSD AIDS AEK (SEQ ID NO: 1), including SHESTEQSDAIDpSAEK (SEQ ID NO: 2), pSHESTEQSDAIDpSAEK (SEQ ID NO: 3) SHEpSTEQSDAIDpSAEK (SEQ ID NO: 4) and pSHEpSTEQSDAEDpSAEK (SEQ ID NO: 5).

In another aspect, the invention features pharmaceutical preparations comprising a phosphopeptide and a pharmaceutically acceptable carrier.

In yet a further aspect, the invention features methods for treating of preventing kidney stone formation or growth, as well as other conditions that result from aberrant calcium oxalate monohydrate (COM) or hydroxyapatite (HA) containing crystals in a subject, comprising administering to the subject an effective amount of an inhibitory phosphopeptide.

Other features and advantages of the invention will be apparent based on the following Detailed Description and Claims. Brief Description of the Drawings

Figure 1 shows Scanning Electron Micrograph (SEM) images of calcium oxalate monohydrate (COM) crystals formed in the presence of : a) no peptide, b) 2 μg/ml of PO (SEQ ID NO. 1), c) 20 μg/ml PO (SEQ ID NO. I)₅ d) 2 μg/ml of Pl (SEQ ID NO. 2), e) 20 μg/ml of Pl (SEQ ID NO. 2), f) 2 μg/ml of P3 (SEQ ID NO. 5, and g) 20 μg/ml of P3 (SEQ ID NO. 5). Pl inhibited crystal growth perpendicular to the {100} faces, P3 inhibited crystal growth perpendicular to both {100} and {120} faces. The degree of inhibition of crystal growth increased with the number of phosphate groups on the peptide.

Figures 2A and 2B show Scanning Electron Micrograph (SEM) images of calcium oxylate (COM) crystals formed in the presence of : A) 2 μg/ml of P2A (SEQ ID NO. 3) and B) 2 μg/ml P2B (SEQ ID NO. 4). Both phosphopeptides inhibited crystal growth perpendicular to the {100} faces. The degree of inhibition of crystal growth increased with the number of phosphate groups on the peptide (i.e. P2A and P2B more effectively inhibited crystal growth as compared to Pl, but less effectively inhibited crystal growth as compared to P3.)

Figure 3 shows scanning confocal fluorescence microscopy images of AlexaFluor- 488-labeled peptides added to preformed COM crystals. Images shown are optical sections taken approximately half-way through the thickness of the crystals. Panels a, b and c are combined red (crystal) and green (peptide) green channel images; panels d, e and fare green-channel images converted to grey scale. Adsorption by PO (SEQ ID NO. 1) is shown in panels a and d. Adsorption by Pl (SEQ ID NO. 2) is shown in panels b and e. Adsorption by P3 (SEQ ID NO. 5) is shown in panels c and f.

Figure 4 provides molecular dynamic simulations of PO (SEQ ID NO. 1), Pl (SEQ ID NO. 2) and P3 (SEQ ID NO. 5) to {100} face of COM: peptide centre-of-mass calculations. The inset shows the conformation of P3 at 15 nsec.

Figure 5 provides molecular dynamic simulations of PO (SEQ ID NO. 1), Pl (SEQ ID NO. 2) and P3 (SEQ ID NO. 5) to {100} face of COM: amino acid centre-of-mass calculations A. z-axis coordinate and B. root mean square deviation from initial position. Detailed Description of the Invention

1. General

The instant inventions are based at least in part on studies of the interaction between COM crystals and the synthetic peptide, PO (SEQ ID NO. 1). To investigate the role of phosphorylation, the peptide has been synthesized in forms containing 0, 1 , 2 or 3 phosphoserines. Scanning confocal microscopy of fluorescence-tagged peptides was used to determine the faces of COM with which the peptides interact. Scanning electron microscopy was used to characterize the effects of these peptides on COM growth habit (crystal size and shape). Finally molecular dynamics was used to simulate the interactions of the peptides with the {100} lattice plane of the COM crystal. These studies have identified face-specific and phosphorylation-dependent interactions between the peptides and COM and provide new insights into the mechanisms by which peptides can inhibit the formation of biologically relevant crystals. Figures 1 and 2 show Scanning Electron Micrograph (SEM) images of calcium oxalate (COM) crystals formed in the presence of no peptide (Figure Ia) and 2 or 20 g/ml of unphosphorylated SEQ ID NO. 1 (Figures Ib and Ic) and certain phosphopeptides (Figures ld-g and 2). Control crystals (no peptide) were penetration twins with {100}, {010} and {121} faces developed (Fig. Ia). Crystals grown in the presence of PO were very similar to controls (Fig. Ib and Ic). Those grown in the presence of Pl were of normal length (<001> directions) and thickness (<010> directions) but were decreased in width (<100>) (Fig. Id and Ie). Growth of COM in the presence of P2A and P2B was less than in the presence of Pl (Fig. 2A and 2B). Growth of COM in the presence of P3 had the most profound effects on growth habit. At 2 μg/ml, crystals were of normal length but reduced in width; no twin axis was apparent and interfacial edges (e.g., {100}/{121} were rounded (Figure If)). At 20 μg/ml P3 crystals were reduced in length as well as width and {100} faces were not apparent (Figure Ig).

To identify the faces of COM with which the peptides interact, aliquots of each of the peptides were labeled with AlexaFluor-488 and added to solutions in which COM crystals had been grown. Images were collected with He/Ne (red) and Kr/ Ar (green) lasers every 0.5-1.0 μm along the microscopic z axis. The images shown in Figure 3 were collected approximately half-way through the thickness of crystals nucleated from an {010} face. For all three peptides, fluorescence was mainly on {100} faces of the crystal. In the case of PO and P3, some fluorescence was also associated with {121} faces. No significant adsorption of PO, Pl or P3 to {010} faces was observed; this was determined by examination of rare crystals nucleated from a {100} face and 3-dimensional reconstructions of crystals nucleated from {010} faces.

Molecular dynamics was used to simulate the interactions between the three OPN peptides and the {100} face of COM. Peptides were initially placed in extended conformations parallel to the crystal face. In all cases, the peptide did not adopt a folded conformation in "solution", but rapidly associated with the crystal face, exhibiting considerable motion until multiple contacts were made. The centre of mass of each peptide was calculated, and its position plotted against simulation time (Figure 4). For both PO and Pl, the y coordinate (distance from top layer of crystal atoms) reached a minimum value of approximately 0.8-1.0 nm, but exhibited considerable fluctuation around this value. P3, in contrast, adsorbed more quickly to the {100} face, achieved a closer approximation to the face (~0.65nm) and exhibited a more stable interaction.

To identify the amino acids involved in the interaction between OPN220-235 and the {100} face of COM, the distance between side-chain centre of mass and the surface layer of the crystal was measured every 0.1 nsec over the period 10-15 nsec. These values were then averaged and plotted against residue number (Figure 5a). For PO, the residues closest to the face were carboxylate-b earing residues at or near the C-terminal end of the molecule (D 12, El 5 and Kl 6). For Pl, E6 was in close contact, as were several residues at the C-terminus (pS13, E15 and K16); residues Q7-D12 inclusive were all relatively distant (>0.8 nm) from the face. For P3, all residues were <0.8 nm from the {100} face. The closest residues were pSl, E3, pS4, E6, D9 and E15 (but not pS13). This analysis suggests that, although phosphate groups are clearly important (compare the position of pSl in P3 with those of Sl in PO and Pl), aspartic and glutamic acid — particularly El 5 — play important roles. The root mean square distances (RMSD) of the side-chains were also calculated (Figure 5b). A low RSMD for an amino acid indicates that its position varies relatively little with time. In general, amino acids located close to the face exhibit little movement, as would be expected. Note, however, that the N- and C-terminal residues are particularly mobile (except when the former is phosphorylated), as is Q7. 2 Definitions As used herein, the following terms and phrases are intended to have the following meanings:

"Adsorption" refers to a noncovalent attachment of an inhibitory phosphopeptide to a crystal, for example, through hydrogen bonding, van der Waal's forces, polar attraction, electrostatic forces (i.e., through ionic bonding), or the like.

"Amino acid" is used herein to refer to natural or synthetic molecules including D or L optical isomers, analogs and petidomimetics. "Antibody" is used herein to refer to binding molecules including immunoglobulins and immunologically active portions thereof, i.e., molecules that contain an antigen binding site, including Fab, Fab', F(ab^*)₂, scFv, Fv, dsFv, diabodies, minibodies, Fd fragments and single chain antibodies (SCAs).

"Comprise" and "comprising" are transitional terms that are used in the inclusive, open sense to indicate that additional elements may be included.

"Conservative amino acid substitution" refers to a replacement of one amino acid with another having a similar side chain as defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic siA "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a natural immunoglobulin can be preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a natural immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity. "Consisting essentially of is a transitional phrase that excludes from the scope of any succeeding recitation any other component, step or procedure, excepting those that are not essential to operability.

The phrase "consisting of," excludes any component, step or procedure not specifically delineated or listed. "Crystal" or "crystalline," as used herein refers to a plurality of atoms or molecules that are physically associated with each other in a regular manner and that involve alignment along one or more axes.

"Degenerate" refers to codons that differ in at least one nucleotide from a reference nucleic acid, but encode the same amino acids as the reference nucleic acid. For example, codons specified by the triplets "UCU", "UCC", "UCA", and "UCG" are degenerate with respect to each other since all four of these codons encode the amino acid serine.

"Ectopic calcification," as used herein, refers to aberrant deposition of calcium within the body. Ectopic calcification is inclusive of the deposition of calcium in renal tubules and urine that results in the formation of primarily calcium oxalate-containing kidney stones.

"Hybridization" refers to the binding of complementary strands of nucleic acid (i.e., sense:antisense strands or probe :target-DNA) to each other through hydrogen bonds, similar to the bonds that naturally occur in chromosomal DNA. Stringency levels used to hybridize a given probe with target-DNA can be readily varied by those of skill in the art. "High stringency hybridization" refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 ⁰C, for example, if a hybrid is not stable in 0.018M NaCl at 65 ⁰C, it will not be stable under high stringency conditions, as contemplated herein. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5 x Denhart's solution, 5 x SSPE, 0.2% SDS at 42 ⁰C, followed by washing in 0.1 x SSPE, and 0.1% SDS at 65 ⁰C.

"Inhibit" or "inhibition," means preventing, retarding, or reversing formation, growth or deposition of a crystal.

"Nucleic acid" is used herein to refer to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single and double-stranded polynucleotides. "Percent identity" or "percent similarity" indicates the degree of sameness between two molecules, e.g. peptides or nucleic acids. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of the number of • identical positions shared by the sequences and the percent homology between two sequences is a function of the number of conserved positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity and/or homology between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and

Wunsch ((1970) J. MoI. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web with the extension gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available on the world wide web with the extension gcg.com), using a NWSgapdna CMP matrix and a gap weight of 40, 50, 60, 70; or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.

The percent identity and/or homology between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The terms "treating" or "treatment," as used herein refers to a reduction in the severity and/or frequency of symptoms

3. Inhibitory phosphopeptides

An inhibitory phosphopeptide as referenced throughout the specification and claims is intended to refer to a peptide (i.e. a complex of at least two amino acids) that comprises at least one phosphate group and is capable of inhibiting the formation or growth of calcium oxalate or hydroxyapatite crystals.

Appropriate inhibitory phosphopeptides may consist of the consensus sequences NNNNTSINNNNNNNNpNNN (SEQ ID NO: 6); N_PNNNNNNNNNNNN_PNNN (SEQ ID NO: 7); NNNN_PNNNNNNNNN_PNNN (SEQ ID NO: 8), or N_PNNN_PNNNNNNNNN_PNNN (SEQ ID NO: 9). The amino acids (N) forming all or a part of a peptide may be any of the twenty conventional, naturally occurring amino acids, i.e., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (T), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y). A phosphopeptide may comprise, 1, 2, 3, 4, 5, 6, or more phosphorylated residues. The phosphorylated residue(s) (-- Np--) may include phosphoserine, phosphotyrosine, or phosphothreonine. The phosphorylated residues may be contiguous and adjacent to acidic amino acids. Phosphopeptides that are comprised of at least about 25% of acidic amino acids (such as aspartic acid or glutamic acid), no more than about 10% basic amino acids (such as lysine or arginine) and at least about 15% phosphorylated amino acids, such as phosphoserine and phosphothreonine appear to better mold themselves to crystal faces and thus may be more effective inhibitors of crystal growth. On the other hand, nonpolar amino acids such as valine and leucine do not seem to affect activity. Certain inhibitory phosphopeptides are phosphorylated forms of

SHESTEQSDAIDSAEK (SEQ TD NO: 1), including SHESTEQSDAIDpSAEK (SEQ ID NO: 2), pSHESTEQSDAIDpSAEK (SEQ ID NO: 3) SHEpSTEQSDAIDpSAEK (SEQ ID NO: 4) and pSHEpSTEQSDAIDpSAEK (SEQ ID NO: 5), where "pS" indicates phosphoserine.

Any amino acid in the above sequences may be replaced by an isomer or analog of a conventional amino acid (e.g., a D-amino acid), non-protein amino acids post- translationally modified amino acids enzymatically modified amino acid, a construct or structure designed to mimic an amino acid (e.g., an α, α-disubstituted amino acid, N-alkyl amino acid, lactic acid, β-alanine, naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, and nor-leucine). Phosphopeptide compounds herein also include compounds wherein the naturally occurring amide --CONH-- linkage is replaced at one or more sites within the peptide backbone with a non-conventional linkage such as an N-substituted amide, ester, thioamide, retropeptide (--NHCO-), retrothioamide (--NHCS-), sulfonamido (--SO₂NH-), and/or peptoid (N-substituted glycine) linkage. Accordingly, phosphopeptide molecules herein include pseudopeptides and peptidomimetics. The phosphopeptides of this invention can be (a) naturally occurring, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of larger molecules, (e) produced by methods resulting from a combination of methods (a) through (d) listed above, or (f) produced by any other means for producing peptides. Preferred inhibitory phosphopeptides include, for example, polypeptides having substantially the same amino acid sequence as any one of SEQ ID NOs: 2-5.

Based on SEQ ID NOS: 2-5, and with use of the assays provided in the following examples, one of skill in the art could identify other phosphopeptides with crystal growth inhibitory activity. For example, a phosphopeptide disclosed herein may be modified to include an addition, deletion or replacement of one or more amino acids. Suitable replacements can include isolated conservative amino acid substititutions, such as replacement of a leucine with isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Further modifications may result in more acidic groups, such as by replacing serine or threonine residues, by negatively charged amino acids, such as glutamic acid, aspartic acid, or gamma- carboxyglutamic acid. The substitution or addition of residues, such as kinase phosphorylation consensus sequences, that can be phosphorylated either in vivo or in vitro is also contemplated. Modifications of residues between the native sites of phosphorylation, such as to beneficially orient the phosphorylated residues to interact with hydroxyapatite or COM or to reduce the distance between phosphorylation sites, are also contemplated. If desired, such as to optimize their functional activity, selectivity, stability or bioavailability, an inhibitory phosphopeptide can be modified, for example, to include D- stereoisomers, non-naturally occurring amino acids, and amino acid analogs and mimetics. Examples of modified amino acids are presented in Sawyer, Peptide Based Drug Design, ACS, Washington (1995) and Gross and Meienhofer, The Peptides: Analysis, Synthesis, Biology, Academic Press, Inc., New York (1983), both of which are incorporated herein by reference.

In order to exhibit appropriate activity, phosphopeptides should consist of at least 5 amino acids, but preferably less than 20 amino acids is preferred for use as a drug. Based on the instant disclosure of particular inhibitory phosphopeptides, one of skill in the art could empirically determine optimally sized phosphopeptides, which may for example comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acid residues.

. Phosphopeptides with modified amino and/or carboxy termini are also envisioned. Amino terminus modifications include methylation (e.g., --NHCH₃ or -N(CHa)₂), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid), adding a benzyloxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxylate functionality defined by RCOO-- or sulfonyl functionality defined by R-SO₂-, where R is selected from alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N-terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the peptide compound. The N-terminus may be acetylated. An N-terminal glycine may be acetylated to yield N-acetylglycine (AcG).

Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the peptides of the invention, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. C-terminal functional groups of the compounds of the present invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

One can replace the naturally occurring side chains of the 20 genetically encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for instance with groups such as alkyl, lower allkyl, cyclic A-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic, hi particular, proline analogues in.which the ring size of the proline residue is changed from 5 members to 4, 6, or 7 members can be employed. Cyclic groups can be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic. Heterocyclic groups preferably contain one or more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of such groups include the furazanyl, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1- piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g. thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. Where a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

The phosphopeptide compounds of the invention may also serve as structural models for non-peptidic compounds with similar biological activity. Those of skill in the art recognize that a variety of techniques are available for constructing compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis [See, Morgan and Gainor (1989) Ann. Rep. Med. Chem. 24:243- 252]. These techniques include replacing the peptide backbone with a backbone composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, and N- methylamino acids.

An inhibitory phosphopeptide can be prepared or obtained by methods known in the art including, for example, purification from an appropriate biological source or by chemical synthesis. In addition to synthesis, inhibitory phosphopeptides can be produced, for example, by enzymatic or chemical cleavage of larger sequences. Methods for enzymatic and chemical cleavage and for purification of the resultant protein fragments are well known in the art (see, for example, Deutscher, Methods in Enzymology, Vol. 182, "Guide to Protein Purification," San Diego: Academic Press, Inc. (1990), which is incorporated herein by reference).

Following synthesis and purification, inhibitory phosphopeptides can be modified in a physiologically relevant manner by, for example, further phosphorylation, acylation or glycosylation, using enzymatic methods known in the art. A kinase that can be used to phosphorylate an inhibitory phosphopeptide at biologically relevant sites is casein kinase II. Other serine-threonine kinases known in the art, such as protein kinase C can also be used to phosphorylate.

An inhibitory phosphopeptide can also be recombinantly expressed by appropriate host cells including, for example, bacterial, yeast, amphibian, avian and mammalian cells, using methods known in the art. Methods for recombinant expression and purification of peptides in various host organisms are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992) and in Ansubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), both of which are incorporated herein by reference.

An isolated nucleic acid encoding a peptide of any of SEQ ID NOs: 1-5 may comprise SEQ ID NO: 10. (SEQ ID NO: 10: TCT CAC GAA TCT ACC GAA CAG TCT GAC GCT ATC GAC TCT GCT GAA AAA), as shown below. S H E S T E Q S D A I D S A E K (SEQ ID

NO . 1)

Ser His GIu Ser Thr GIu GIn Ser Asp Ala lie Asp Ser Ala GIu Lys length

= 16

TCT CAC GAA TCT ACC GAA CAG TCT GAC GCT ATC GAC TCT GCT GAA AAA length = 48

Further provided are nucleic acids encoding phosphopeptides that, by virtue of the degeneracy of the genetic code, do not necessarily hybridize to the invention nucleic acids under specified hybridization conditions. Preferred nucleic acids encoding the phosphopeptides are comprised of nucleotides that encode substantially the same amino acid sequence as set forth in SEQ ID NOs: 2-5. Peptide libraries spanning overlapping sequences can be produced using methods known in the art and screened for their functional activity as described herein. 4. Activity Assays

In addition to the assays described in the example, the ability of an inhibitory phosphopeptide selected and prepared as described above to inhibit COM or HA (See e.g., Pampena, D. et al._s (2004) Biochem. J. 378: 1083-1087) crystal formation or growth can be assayed by a variety of in vitro and in vivo assays known in the art or described herein.) For example, cultured vascular cells, such as bovine aortic smooth muscle cells, form calcified deposits in a time-dependent manner when treated with calcification medium containing β-glycerophosphate. Additionally, human vascular smooth muscle cells form calcified deposits in the presence of elevated levels of inorganic phosphate. Other culture systems for assaying the efficacy of an inhibitory phosphopeptide in inhibiting ectopic calcification can be determined by those skilled in the art. For example, an inhibitory phosphopeptide can be assayed using cells or tissues derived from other sites in the body where ectopic calcification occurs including, for example, viscera, skin, and endothelial cells. The amount or extent of ectopic calcification prior to and following administering an inhibitory phosphopeptide can be determined using such culture systems, either qualitatively by a visual or histochemical assessment, or by more quantitative methods. For example, calcified deposits can be detected visually as opaque areas by light microscopy and as black areas by von Kossa staining. The amount or extent of ectopic calcification can also be quantitatively assessed by the method described by Jono et al., Arterioscler.

Thromb. Vase. Biol. 17: 1135-1142 (1997), incorporated herein by reference, or by using a commercially available colorimetric kit such as the Calcium Kit available from Sigma. Alternatively, the amount or extent of ectopic calcification can also be quantitatively assessed using known methods of atomic absorption spectroscopy. The ability of an inhibitory phosphopeptide to inhibit ectopic calcification can also be tested in animal models known in the art to be reliable indicators of the corresponding human pathology. For example, ectopic calcification can be induced by the subcutaneous or circulatory implantation of bioprosthetic valves, such as porcine or bovine valves, into animals. A reduction in the amount or rate of valve calcification by administration of an inhibitory phosphopeptide can be detected, and is a measure of the functional activity of the preparation.

Similarly, animal models that are reliable indicators of human atherosclerosis, age- related vascular calcification and other conditions associated with ectopic calcification are known in the art. For example topical and systemic calciphylaxis, calcinosis and calcergy, which are experimental models of ectopic calcification are described, for example, in Bargmaπn, J. Rheumatology 22:5-6 (1995), Lian et al., Calcified Tissue International, 35:555-561 (1983) and Boivin et al., Cell and Tissue Res. 247:525-532 (1987). An experimental model of calcification of the vessel wall is described, for example, by Yamaguchi et al., Exp. Path. 25:185-190 (1984).

Medical imaging techniques known in the art, such as magnetic resonance imaging, X-ray imaging, computed tomography and ultrasonography, can be used to assess the efficacy of an inhibitory phosphopeptide in inhibiting ectopic calcification in either a human or an animal. For example, the presence and extent of calcium deposits within vessels can be determined by the intravascular ultrasound imaging method described by Fitzgerald et al., Circulation 86:64-70 (1994), incorporated herein by reference. A decrease in the amount or extent of ectopic calcification can readily be identified and is indicative of the therapeutic efficacy of an inhibitory phosphopeptide.

5. Formulations

Administration of inhibitory phosphopeptides can be achieved using various formulations. If desired, inhibitory phosphopeptides can be administered as a solution or suspension together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be, for example, water, sodium phosphate buffer, phosphate buffered saline, normal saline or Ringer's solution or other physiologically buffered saline, or other solvent or vehicle such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester.

A pharmaceutically acceptable carrier can additionally contain physiologically acceptable compounds that act, for example, to stabilize or increase the absorption of the inhibitory phosphopeptide to be administered. Such physiologically acceptable compounds include, for example, carbohydrates such as glucose, sucrose or dextrans; antioxidants such as ascorbic acid or glutathione; chelating agents' such as EDTA, which disrupts microbial membranes; divalent metal ions such as calcium or magnesium; low molecular weight proteins; lipids or liposomes; or other stabilizers or excipients. Inhibitory phosphopeptides can also be formulated with a material such as a biodegradable polymer or a micropump that provides for controlled slow release of the molecule. Additionally, inhibitory phosphohopeptides can be formulated with a molecule, such as a phosphatase inhibitor, that -reduces or inhibits dephosphorylation of the inhibitory phosphopeptide.

Inhibitory phosphόpeptides can also be expressed from cells that have been genetically modified to express the protein. Expression of an inhibitory phosphopeptide from a genetically modified cell provides the advantage that sustained localized or systemic expression of the protein can occur, thus obviating the need for repeated administrations.

Methods for recombinantly expressing proteins in a variety of mammalian cells for therapeutic purposes are known in the art and may be used to administer an inhibitory phosphopeptide. These are described, for example, in Lee et al., Transfusion Medicine II 9:91-113 (1995), which is incorporated herein by reference. Types of cells that are particularly amenable to genetic manipulation which may be used in conjunction with the methods provided herein include, for example, hematopoietic stem cells, hepatocytes, vascular endothelial cells, keratinocytes; myoblasts, fibroblasts and lymphocytes.

A nucleic acid encoding an inhibitory phosphopeptide can be operatively linked to a promoter sequence, which can provide constitutive or, if desired, inducible expression of appropriate levels of the encoded inhibitory phosphopeptide. Suitable promoter sequences for a particular application of the method can be determined by those skilled in the art and will depend, for example, on the cell type and the desired inhibitory phosphopeptide expression level.

The nucleic acid encoding an inhibitory phosphopeptide can be inserted into a mammalian expression vector and introduced into cells by a variety of methods known in the art (see, for example, Sambrook et al., 1989; and Ausubel et al., 1994). Such methods include, for example, transfection, lipofection, electroporation and infection with recombinant vectors. Infection with viral vectors such as retrovirus, adenovirus or adenovirus-associated vectors is particularly useful for genetically modifying a cell. A nucleic acid molecule also can be introduced into a cell using known methods that do not require the initial introduction of the nucleic acid sequence into a vector.

In one embodiment of the invention, a prosthetic device can be contacted with an inhibitory phosphopeptide. Contacting a prosthetic device with an inhibitory phosphopeptide will effectively prevent or reduce ectopic calcification of the prosthetic device, preventing failure of the device and the need for premature replacement. The prosthetic device can be contacted with an inhibitory phosphopeptide either prior to, during or following implantation into an individual, as needed.

An inhibitory phosphopeptide can contact a prosthetic device by attaching the molecule either covalently or non-covalently to the prosthetic device. An appropriate attachment method for a particular application of the method can be determined by those skilled in the art. Those skilled in the art know that an appropriate attachment method is compatible with implantation of the prosthetic device in humans and, accordingly, will not cause unacceptable toxicity or immunological rejection. Additionally, an appropriate attachment method will enhance or not significantly reduce the ability of an inhibitory phosphopeptide to inhibit ectopic calcification of the prosthetic device and the surrounding tissue.

Methods for covalently attaching proteins to polymers, metals and tissues are known in the art. For example, an inhibitory phosphopeptide can be attached to the prosthetic device using chemical cross-linking. Chemical cross-linking agents include, for example, ^' glutaraldehyde and other aldehydes. Cross-linking agents that link an inhibitory phosphopeptide to a prosthetic device through either a reactive amino acid group, a carbohydrate moiety, or an added synthetic moiety are known in the art. Such agents and methods are described, for example, in Hermason, Bioconjugate Techniques, Academic

Press. San Diego (1996), which is incorporated herein by reference. These methods can be used to contact a prosthetic device with a therapeutically effective amount of an inhibitory phosphopeptide.

An inhibitory phosphopeptide can also be attached non-covalently to the prosthetic device by, for example, adsorption to the surface of the prosthetic device. A solution or suspension containing an inhibitory phosphopeptide, together with a pharmaceutically acceptable carrier, if desired, can be coated onto the prosthetic device in a therapeutically effective amount.

To provide sustained delivery of an inhibitory phosphopeptide, a prosthetic device can also be contacted with an inhibitory phosphopeptide produced by cells attached to the prosthetic device. Such cells can be seeded onto the prosthetic device and expanded either ex vivo or in vivo. Appropriate cells include cells that normally produce and secrete an inhibitory phosphopeptide including, for example, macrophages, smooth muscle cells or endothelial cells. Additionally, cells that have been genetically modified to produce an inhibitory phosphopeptide including, for example, endothelial cells and fibroblasts, can be attached to the prosthetic device. The cells that are attached to the prosthetic device are preferably either derived from the individual receiving the prosthetic implant, or from an immunologically matched individual to reduce the likelihood of rejection of the implant. The ability of an inhibitory phosphopeptide that contacts a prosthetic device to inhibit ectopic calcification can be determined by various methods known in the art. One such method is to implant the prosthetic device into animals and measure calcium deposition, in response to administration of an inhibitory phosphopeptide. Either a decrease in the rate or the amount of calcium deposition at the site of the explant is indicative of the therapeutic efficacy of the composition.

6. Methods of Use

By inhibiting growth of aberrant COM or HA containing crystal growth, the phosphopeptide described herein should prove useful for treating or preventing diseases or conditions associated with aberrant or ectopic calcification, such as kidney stone formation. For example, ectopic calcification can result from inflammation or damage to the affected tissues or can result from a systemic mineral imbalance. Commonly, ectopic calcification occurs in vascular tissue, including arteries, veins, capillaries, valves and sinuses. Inflammation or damage to the blood vessels can occur, for example, as a result of environmental factors such as smoking and high-fat diet. Inflammation or damage can also occur as a result of trauma to the vessels that results from injury, vascular surgery, heart surgery or angioplasty. Vascular calcification is also associated with aging and with disease, including hypertension, atherosclerosis, diabetes, renal failure and subsequent dialysis, stenosis and restenosis.

Ectopic calcification also occurs in non-vascular tissues, such as tendons (Riley et al., Ann. Rheum. Dis. 55:109-115 (1996)), skin (Evans et al., Pediatric Dermatology 12:307-310 (1997)), sclera (Daicker et al., Opthalmologica 210:223-228 (1996) and myometrium (McCluggage et al., Int. J. Gynecol. Pathol. 15:82-84 (1996)). In diseases resulting in systemic mineral imbalance, such as renal failure and diabetes, ectopic calcification in visceral organs, including the lung, heart, kidney and stomach, is common (Hsu, Amer. J. Kidney Disease 4:641-649 (1997), incorporated herein by reference). Furthermore, ectopic calcification is a frequent complication of the implantation of biomaterials, prostheses and medical devices, including, for example, bioprosthetic heart valves (Vyavahare et al., Cardiovascular Pathology 6:219-229 (1997), incorporated herein by reference). The methods provided herein are applicable to ectopic calcification that occurs in association with all of these conditions, as well as, for example, heart or circulatory diseases such as Arteriosclerosis, Atherosclerosis, Valve Calcifications, Calcific Aortic Stenosis, Vascular Thrombosis; Dental Diseases such as Dental Calculus (dental pulp stones), calcification of the dentinal papilla, and Salivary Gland Stones, Kidney and Bladder Stones, Gall Stones, Gout, Pancreatic Duct Stones, Adrenal Calcification, Liver Cysts, Chronic Calculous Prostatitis, Prostate Calcification, Calcification in Hemodialysis Patients, Polycystic Kidney Disease, Glomerulopathies; Eye Diseases such as Corneal Calcifications, Cataracts, Ear Diseases such as Otosclerosis, Thyroglossal cysts, Thyroid Cysts, Ovarian Cysts, Skin diseases such as Calcinosis Cutis, Skin Stones, Calciphylaxis, Choroid Plexus Calcification, Neuronal Calcification, Calcification of the FaIx Cerebri, Calcification of the Intervertebral Cartilage or Disc, Calcific Tenditis, calcification of articular cartilage, synovium and synovial fluid in Osteoarthritis, Gout, Pseudogout or Chondrocalcinosis, Bone Spurs, Diffuse Interstitial Skeletal Hyperostosis, Bronchial Stones, Calcifications and Encrustations of Implants, Mixed Calcified Biofϊlms.

The methods of the invention can advantageously be used to prevent ectopic calcification of prosthetic heart valves, such as an aortic or atrioventricular valve, with or without a stent. Replacement heart valves can be made of a variety of materials, including metals, polymers and biological tissues, or any combination of these materials. Bioprosthetic valves include xenografted replacement valves from mammals, such as ovine, bovine and porcine, as well as human valves. Bioprosthetic heart valves are commonly subjected to tissue fixation and can additionally be devitalized prior to implantation. Inhibitory phosphopeptides can be administered to an individual in a therapeutically effective amount to inhibit ectopic calcification. Appropriate formulations, dosages and routes of delivery for administering an inhibitory phosphopeptide are well known to those skilled in the art and can be determined for human patients, for example, from animal models as described previously. The dosage of an inhibitory phosphopeptide required to be therapeutically effective can depend, for example, on such factors as the extent of calcification, the site of calcification, the route and form of administration, the bio-active half-life of the molecule being administered, the weight and condition of the individual, and previous or concurrent therapies. The appropriate amount considered to be a therapeutically effective dose for a particular application of the method can be determined by those skilled in the art, using the guidance provided herein. One skilled in the art will recognize that the condition of the patient needs to be monitored throughout the course of therapy and that the amount of the composition that is administered can be adjusted accordingly.

For treating humans, a therapeutically effective amount of an inhibitory phosphopeptide is to be administered, which is any amount deemed nontoxic but sufficient to inhibit biomineralization. If an inhibitory phosphopeptide is administered several times a day, or once a day, or once every several days, a lower dose would be needed than if an inhibitory phosphopeptide were administered only once, or once a week, or once every several weeks. Similarly, formulations that allow for timed-release of an inhibitory phosphopeptide would provide for the continuous release of a smaller amount than would be administered as a single bolus dose.

Inhibitory phosphopeptides can be delivered systemically, such as intravenously or intraarterially, to inhibit ectopic calcification throughout the body. Inhibitory phosphopeptides can also be administered locally at a site known to contain or predicted to develop ectopic calcification. Such a site can be, for example, an atherosclerotic plaque, a segment of artery undergoing angioplasty or the site of prosthetic implantation. Appropriate sites for administration of an inhibitory phosphopeptide can be determined by those skilled in the art depending on the clinical indications of the individual being treated and whether or not the individual is concurrently undergoing invasive surgery.

Incorporation by Reference

All publications, patents and patent applications referred to herein are hereby expressly incorporated by reference in their entirety .

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to.be encompassed by the following claims

Exemplification

The inventions having been generally described may be more readily understood by reference to the following examples.

Control of calcium oxalate crystal growth by face-specific adsorption of an osteopoπtin phosphopeptide Materials and Methods

Peptides: The following peptides were synthesized manually using Fmoc chemistry as previously described (Pampena, D. A. et al., (2004) Biochem J. 378: 1083-1087) and studied in all experiments:

PO: SHESTEQSDAIDSAEK (SEQ ID NO. 1 ) → 0 phosphate groups

Pl: SHESTEQSDAIDpSAEK (SEQ ID NO. 2) → 1 phosphate group

P2A: pSHESTEQSDAIDpSAEK (SEQ ID NO. 3) -→ 2 phosphate groups P2B: SHEpSTEQSDAIDpSAEK (SEQ ID NO. 4)→ 2 phosphate groups

P3: pSHEpSTEQSDAIDpSAEK (SEQ ID NO. 5) -→ 3 phosphate groups

Residue "pS" indicates phosphoserine.

Fluorescence labeling and solution preparation of polypeptides: Peptides were labeled with the Alexa fluorochrome according to the manufacturer's recommendations. Briefly, 5 μl of AlexaFluor-488 carboxylic acid (AlexFluor-488 Invitrogen Corp) in dimethylformamide (10 μg/μl; HPLC grade, 99.9% was added to 200 μl of peptide in phosphate-buffered saline (2.5 mg/ml) and 20 μl of 1 M disodium carbonate (Na₂COa), pH ~8.3, and incubated for 1 h at room temperature (23°C). Unconjugated label was removed by extensive dialysis against Tris-buffered saline, pH 7.4, using 1-kDa dialysis tubing (Spectra/Por 3, SPECTRUM LABORATORIES, Rancho Dominques, CA). After freeze- drying, the samples were stored at -20°C. All chemicals were supplied by SIGMA- ALDRICH LTD. (Oakville, Canada) except Na₂CO₃, which was purchased from MERCK KgaA {Damrstadt, Germany). Amino acid analysis (Alberta Peptide Institute; University of Alberta, Edmonton, Canada) was carried out using norleucine as an internal standard to determine the yield of labeled peptides. The masses obtained were used to prepare aqueous stock solutions peptides. In addition, aqueous stock solutions of 2 mg/ml and 20 μg/ml unlabelled peptide were prepared.

Crystallization experiments: Crystallization of COM was initiated using the method previously described (Grohe, Bernd et al., (2006) J. Crystal Growth 295: 148-157). Final concentrations were 1 mM calcium nitrate, 1 mM sodium oxalate, 10 mM sodium acetate and 150 mM sodium chloride. For scanning electron microscopy, 1-ml aliquots of these solutions were added to wells of tissue-culture plates (24-well), FALCON, Becton Dickinson; Franklin Lakes, NJ) containing freshly cleaved mica disks (diameter: 9.5 mm, V-I grade, SPI SUPPLIES, Toronto, Canada). If unlabeled peptide was added to the wells, the volume of water was correspondingly reduced. Following incubation (BAXTER Scientific PRODUCTS, Ultra Tec WJ 501 S; Mississauga, Canada) at 37°C for 30 min, the mica disks were rinsed with deionized water and air-dried. The final concentration of peptide used was either 2 or 20 μg/ml.

For confocal microscopy, 200-μl aliquots of calcium oxalate solutions were added to glass-bottomed polystyrene dishes (35 mm diameter; glass-bottom: grade no. 1.5, diameter: 10 mm; MATTEK CORPORATION, Ashland, MA). Each dish was covered with laboratory film (PARAFILM "M", Chicago, IL) to prevent evaporation and incubated at room temperature for 3 h. The dish was then placed on the heated (37°C) stage of a Zeiss LSM 410 confocal microscope (CARL ZEISS, Germany) and a 20 μl aliquot of a 0.1 or 0.2 μg/ml solution of peptide was added to the solution. The peptide was allowed to adsorb to the crystals for a period of 45 min prior to imaging.

Scanning electron microscopy: A LEO 1540XB scanning electron microscope equipped with a Gemini field emission column was employed. Precipitates on mica substrates were investigated without metal coating, using an acceleration voltage of 1 kV and a working distance of 3.5 mm. SEM images were imported into Corel DRAW x3 (Corel

Corporation), cropped and indexed and the final images exported as bitmaps. All images reported are unfiltered data.

Scanning confocal microscopy Scanning confocal interference microscopy (SCIM; (Grohe, W.K. et al. (2006) J.Crystal Growth 295:148-157) was used to image the crystal- glass interface. Conventional scanning confocal microscopy (SCM) was used to make optical sections at higher levels of the crystal and to image fluorescence-labeled peptides. In both cases, a 63X oil-immersion objective and a 90/10 mirror as a beam-splitter were used. All procedures were carried out in the dark to avoid the effects of scattered light on crystal imaging and to prevent the fluorochrome from bleaching. Prior to the first scan of every crystal, pre-alignments of the microscope were carried out and the focus adjusted to the interface between, crystal and glass. If required, the focus was adjusted again during image acquisition. Crystals were scanned using a helium/neon laser (• »=632.8 ran). To excite the fluorochrom, a krypton/argon laser (• «=488 nm) was used. Images of crystals were obtained in a single scan (4 sec) with the gain and offset selected to provide optimal contrast of the crystal. The pinhole was adjusted to less than 1 airy unit to provide maximum resolution. Fluorescence images were obtained using an LP 515 emission filter . (the emission maximum for AlexaFluor-488 is at 519nm). Gain and offset were adjusted to provide optimal contrast, and the pinhole was set to maximum aperture. Ten large (~20m x 5 μm), well-formed COM crystals nucleated from a {010} face were scanned in optical sections every 500 nm along the z-axis (corresponding to the "010"directions of the crystal). The final concentration of peptide was either 0.1 or 0.2 μg/ml.

Molecular dynamics: Atomic scale simulations were performed using the GROMACS suite. The coordinates for the COM {100} face were taken from previously obtained experimental results. The .topology for oxalate was generated using PRODRG. Extended conformations were used as the initial peptide structure. For each simulation, peptides were oriented parallel to the crystal surface where the center of mass difference between the crystal slab and the peptide was approximately 4 nm in the direction perpendicular to the surface. The crystal slab was placed at the center of the periodic cell and constructed to be approximately 0.7 nm thick with the Ca²⁺ dense layers of the {100} face exposed on each side. Three-dimensional periodic boundary conditions were defined with the size of the periodic cell being 8.7 nm x 6.2 nm in the plane of the surface and 10 nm perpendicular to the surface. The system was solvated with simple point charge (SPC) water and CF counter-ions were added to maintain a system net charge of zero. Energy minimization was performed without constraints using the steepest descent integrator for 1000 steps with an initial step size of 0.1 A.

Molecular dynamics simulations were performed using the NVT ensemble at 300 K. Crystal atoms were assigned as freeze groups to restrict movement. A Berendsen thermostat with a coupling time constant of 0.2 ps was employed and the particle mesh Ewald method was used for electrostatics. The time step was set to 2 fs. Systems were simulated for 15 ns each. All simulations were run in parallel over eight processors on SHARCNET.

Center of mass and root mean square deviation analysis were performed using the g_traj and g_rms tools of the GROMACS suite, respectively. Results

Scanning Cόnfocal Imaging: PO exhibited weak fluorescence, mainly on {100} faces of the crystal. The fluorescence associated with Pl was more intense, and was very specifically associated with {100} faces. Addition of P3 resulted in less crystal-associated fluorescence than Pl, possibly because the higher acidity of P3 reduced the efficiency of labeling with AlexaFluor. Nonetheless, the images indicate that P3 is less specific than Pl for {100} faces, as significant P3 fluorescence is associated with {121} faces. For all three peptides, there was no significant fluorescence associated with {010} faces of the crystal. SEM Imaging: Crystals grown in the presence of peptide PO, at either 2 or 20 μg/ml, were very similar to controls (no peptide). Those grown in the presence of Pl were of normal length (<001> directions) and thickness (<010> directions) but were decreased in width. Growth of COM in the presence of P3 had the most profound effects on growth habit. At 2 μg/ml, crystals were of normal length but reduced in width; no twin axis was apparent and interfacial edges were rounded. At 20 μg/ml P3, crystals were reduced in length as well as width and {010} faces were not apparent.

Molecular Dynamics: In all cases, the peptide rapidly associated with the crystal face, exhibiting considerable motion until multiple contacts were made between phosphate and carboxylate groups (side-chain and C-terminus) of the peptide and Ca²⁺ ions of the crystal. The conformation of P3 at 15 nsec of simulation time was measured and the centre of mass of each peptide was calculated, and its position plotted against simulation time. For both PO and Pl, they coordinate (distance from crystal surface) reached a minimum value of approximately 0.8-1.0 nm, but exhibited considerable fluctuation around this value. P3, in contrast, adsorbed more quickly to the {100} face, achieved a closer approximation to the face (y * 5.9 nm) and exhibited a more stable interaction.

Claims

1. A phosphopeptide selected from the group consisting of: SEQ ID NOs: 6-9, wherein said phosphopeptide is capable of inhibiting calcium oxalate crystal growth.

2. A phosphopeptide of claim 1 , which is SEQ ID NO. 2.

3. A phosphopeptide of claim 1 , which is SEQ ID NO. 3.

4. A phosphopeptide of claim 1, which is SEQ ED NO. 4.

5. A phosphopeptide of claim 1, which is SEQ ID NO. 5.

6. A phosphopeptide of claim 1 , wherein the phosphopeptide is capable of inhibiting hydroxyapatite crystal growth.

7. A pharmaceutical composition comprising an effective amount of a phosphopeptide of claim 1 and a pharmaceutically acceptable carrier.

8. A phosphopeptide, which consists of 10-20 amino acids, wherein at least 25% of the amino acids are acidic, no more than 10% of the amino acids are basic and at least 15% of the amino acids are phosphorylated and the remaining amino acids are neutral amino acids, wherein the phosphopeptide is not PQNSVpSpSEETDD (SEQ DD NO. 11), SSHELpSSEVpSNE (SEQ ID NO. 12), pSSHELpSSEVpSNE (SEQ ID NO. 13).

9. A phosphopeptide of claim 8, wherein the acidic amino acids are selected from the group consisting of: aspartic acid, glutamic acid or gamma carboxyglutamic acid.

10. A phosphopeptide of claim 8, wherein the basic amino acids are selected from the group consisting of lysine or arginine.

11. A phosphopeptide of claim 8, wherein the phosphorylated amino acids are selected from the group consisting of: phosphoserine, phosphothreonine and phosphotyrosine.

12. A pharmaceutical composition comprising an effective amount of a phosphopeptide of claim 8 and a pharmaceutically acceptable carrier.

13. A phosphopeptide that contains no more than 20 amino acids and is 90% identical to SEQ ID NO. 2.

14. A pharmaceutical composition comprising an effective amount of a phosphopeptide of claim 13 and a pharmaceutically acceptable carrier.

15. A phosphopeptide that contains no more than 20 amino acids and is 90% identical to SEQ ID NO. 3.

16. A pharmaceutical composition comprising an effective amount of a phosphopeptide of claim 15 and a pharmaceutically acceptable carrier.

17. A phosphopeptide that contains no more than 20 amino acids and is 90% identical to SEQ ID NO. 4.

18. A pharmaceutical composition comprising an effective amount of a phosphopeptide of claim 17 and a pharmaceutically acceptable carrier.

19. A phosphopeptide that contains no more than 20 amino acids and is 90% identical to SEQ ID NO. 5.

20. A pharmaceutical composition comprising an effective amount of a phosphopeptide of claim 19 and a pharmaceutically acceptable carrier.

21. A method for inhibiting calcium oxalate monohydrate crystal growth comprising coating the crystal with an appropriate amount of a phosphopeptide selected from the group consisting of SEQ ID NOs: 6-9.

22. A method for treating or preventing kidney stone formation or growth in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of claim 7.