JP2002519304A - Novel Staphylococcus Peptides for Bacterial Interference - Google Patents

Abstract

(57) [Summary] The present invention provides a cyclic peptide having the following constitution. Where X is selected from the group of amino acids, amino acid analogs, peptide analogs and non-amide isomers, Z is selected from the group of synthetic amino acids and biosynthetic amino acids, and R is oxygen, nitrogen and carbon Where n is a number from 0 to 10, and y is a number from 1 to 10.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Throughout this application, various publications are referenced by author and date. All citations for these documents appear in alphabetical order immediately after the sequence listing and claims at the end of the specification. The disclosures of these documents are incorporated herein by reference in their entirety to describe the state of the art in more detail. The sequence listing is provided after the reference listing and before the claims.

[0002]

TECHNICAL FIELD OF THE INVENTION

 The present invention relates generally to synthetic cyclic peptides for bacterial interference.

[0003]

[Background Art]

Staphylococcus aureus (S. aureus) is an important pathogen in humans whose risk of developing antibiotic resistance to currently available treatments is currently increasing. As a result, there is an urgent need to identify new classes of antibiotics that are effective against these drug-resistant bacterial strains. The phenomenon of "bacterial interference" could be an unexplored avenue for designing these new treatments. Bacterial interference refers to the ability of one organism to interfere with the biological function of another organism. Until recently, this survival process was thought to occur solely by growth arrest mechanisms (Ji, G., et al., 1997).
However, S. et al. Involved in blocking the so-called agr response. Aureus (S. aure)
s) has been described by a novel class of bacteria (Novic
k, R. P. ) Et al., 1993; Morfeldt, E. et al.
995). This process is mediated by a short secreted peptide containing a putative thiololactone ring structure. Chemical synthesis has confirmed that the native Agr peptide contains a thiololactone moiety and that this structure is absolutely necessary for full biological activity. Structure-activity studies have also been described by the present invention providing insight into the nature of the agr activation and inhibition mechanisms.

[0004] Accessory genes allow bacteria to survive and multiply in plant or animal hosts. S. In S. aures, these virulence factors (cytotoxic and tissue destructive enzymes) are two different promoters, P2 and P3.
Under the control of the agr locus containing RNA transcripts of the P3 promoter are responsible for up-regulation of secreted virulence factors and down-regulation of surface proteins, agr response (Novick, RP et al.
1993, Morfeldt, E. et al., 1995). There are four genes, agrA-D, in the P2 operon encoding the cytosolic, transmembrane and extracellular components of the density-sensitive autoinduction circuit (Novick,
R. P. Et al., 1995). The product of the agrD gene is a propeptide, a complete membrane protein, that is processed and secreted by AgrB. The active AgrD peptide is then believed to bind to the transmembrane receptor encoded by the agrC gene. Binding of the AgrD peptide triggers a standard two-component signaling pathway in which the AgrC receptor is autophosphorylated on histidine residues and subsequently trans-phosphorylated on the AgrA gene product. The phosphorylated AgrA then activates the transcription of the P2 and P3agr promoters (Novik (Novich)
ck), R.C. P. Et al., 1995).

[0005] S. aures strains can be divided into at least three groups (Ji, G., et al., 1997), each of which secreted AgrD peptide activates an agr response within the same group, Agr response of strains belonging to other groups can be inhibited. It is the latter effect that constitutes a novel form of bacterial interference (di (
Ji, G .; ) Et al., 1997). The AgrD self-inducing peptide formed by treatment and secretion with AgrB consists of 7-9 residues. Interestingly, the sequence is highly variable between groups, but all contain 5 amino acids of a conserved C-terminal cysteine residue. Analysis of the AgrD peptide isolated from the cell supernatant by mass spectrometry showed a mass discrepancy of -18 Da compared to the mass predicted based on the peptide sequence (Ji, G., et al.). , 1995). Taken together with this observation, the presence of cysteine residues conserved in the AgrD peptides indicates that these secretory peptides contain an intramolecular thiol ester bond between the cysteine sulfhydryl group and the carboxy-terminus. (Ji, G.)
Et al., 1997). Purified AgrD consistent with the thiololactone structure
The addition of hydroxylamine to the peptide was observed to abolish biological activity (Di (Gi, G.) et al., 1997).

[0006] The inability to isolate large quantities of secreted AgrD peptide is attributed to Agr
This means that little is known about the biochemistry of the D / AgrC interaction. For example, it activates the agr response (S. aureus of the same group)
es) The potency of the AgrD peptide in inhibiting (in strains) or inhibiting (in another group of S. aures) is unknown. Similarly, it is necessary to determine whether the putative thiololactone structure in the AgrD peptide is required for activating agr response, inhibiting agr response, or both. The present disclosure provides such an elucidation. This study, described in detail herein, confirms the presence of a thiololactone moiety within the Agr peptide by total chemical synthesis. Bringing this system closer to synthesis addresses more vigorous biochemical and structure-activity studies on the AgrD / AgrC interaction. The disclosure of the present invention further states that the elimination of the thiol ester component of the cyclic structure can activate the agr response while at the same time destroying the action of preserving (and enhancing) the inhibitory action.

[0007]

DISCLOSURE OF THE INVENTION

 The present invention provides a structure,

[0008]

Embedded image Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres, Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, and R is oxygen, nitrogen and carbon Is selected from the group consisting of
And y is 1-10)

There is provided a cyclic peptide comprising: The invention also contemplates a peptide composition comprising the provided cyclic peptide and a carrier.

[0010] The present invention _also provides cyclic peptide (in the formula, including an annular bond other than a thioester with _{NH 2 -X (n) -Z-} X (y) -COOH amino acid sequence Z residue and COOH of, X Is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres; Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids; n is 0-10; 10). The present invention also treats infectious diseases comprising administration of a composition comprising the provided cyclic peptide and a carrier, and a pharmaceutical composition made and which may be made according to the teachings of the invention herein. To provide a treatment method. Still further, the invention extends to a method for producing a cyclic peptide comprising the cyclization protocol described in further detail herein and exemplified in Example 1 and FIG. 1A, which is itself an invention.

Accordingly, a primary object of the present invention is to provide NH ₂ -X _(n) -ZX _(y) -COOH
Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres; Is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, wherein n is 0-10 and y is 1-10).

Still another object of the present invention is to eliminate the activities action, holds the inhibitory _effect, and the amino acid sequence and the Z residue and COOH of _{NH 2 -X (n) -Z-} X (y) -COOH Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres, and Z is the group consisting of synthetic amino acids and biosynthetic amino acids Wherein n is 0-10 and y is 1-10).

Yet another object of the present invention is to provide NH ₂ —X _(n) —ZX _(y) —COOH
And a cyclic bond other than the thioester of the Z residue and COOH,
A cyclic peptide whose bond is selected from the group consisting of a lactam ring and a lactone ring (
Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres, Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, n is 0-10, y is from 1 to 10).

[0014] Yet another object of the present invention is to provide a pharmaceutical composition comprising the provided peptide and a pharmaceutically acceptable carrier.

[0015] Yet another object of the present invention is to provide a subject with S. cerevisiae. Aureus (S. aures)
Administering to the subject an effective amount of the pharmaceutical composition provided to treat the infectious disease. It is to provide a method for treating S. aures infection.

Yet another object of the present invention is to provide a method for producing a cyclic peptide herein, comprising a solid phase cyclization protocol exemplified and described herein.

Other objects and advantages will become apparent to those skilled in the art by reference to the following description, which proceeds with reference to the following illustrative figures.

[0018]

[Detailed description]

 The present invention provides a structure,

[0019]

Embedded image Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres, Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, and R is oxygen, nitrogen and carbon Is selected from the group consisting of
And y is 1-10)

There is provided a cyclic peptide comprising:

The present invention also provides a cyclic peptide containing an amino acid sequence of NH ₂ -X _(n) -ZX _(y) -COOH and a cyclic bond other than a thioester between Z residue and COOH (wherein X Is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres; Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids; n is 0-10; 10).

One embodiment of the present invention is a compound comprising a provided peptide and a substance for the peptide or a polymer thereof.

[0023] Yet another embodiment of the present invention comprises assembling the linear peptide component being manufactured on the PEGA resin support to form a protected, attached peptide chain;
Treating and deprotecting the resulting peptide chain, and cleaving the deprotected peptide from the solid support with a buffer at neutral pH to form the desired cyclic peptide. And recovering the cyclic peptide. The method for producing a cyclic peptide of the present invention comprises the steps of: More specifically, in the method of the present invention, a linear peptide chain having a composition corresponding to the cyclic peptide is converted to 3-mercapto-propionamide-polyethylene glycol-poly- (N ₁ N ₄ -dimethacrylamide) (HS- Assembling on a solid phase resin support containing PEGA) to form a protected peptide assembly, and treating the protected peptide assembly of the previous step to deprotect said peptide assembly Treating the deprotected peptide with a buffered aqueous solution at a pH of about 7.0 for a period of time sufficient to form the cyclic peptide and cleave the peptide from the solid phase resin support; Recovering the cyclic peptide.

As described in Example 1 and illustrated in FIG. 1A, the method of the present invention uses a solid phase cyclization protocol as the last step to form a cyclic peptide of the present invention. Also,
The method of the invention involved the initial production of a peptide that was completely deprotected on a solid support via a reactive thiol ester linkage. Thus, a typical solid phase resin support suitable for use in the method of the present invention is BOC-AA- (chain peptide assembly) -P
EGA may be included. Further, the deprotection treatment following assembly of the peptide on the resin support can be performed, for example, with HF for about 1 hour. Thereafter, cleavage of the peptide from the support and formation of the cyclic peptide can be performed with a buffer such as Na ₂ PO ₄ and acetonitride. Also, this step is performed for a time sufficient to effect cleavage and cyclization, and may range, for example, for about 12 hours. In general, the above reagents and processing parameters may be varied within the scope of the invention, and the invention is intended to include such modifications within the spirit and scope of the invention.

In another embodiment of the invention, the cyclic peptide is capable of inhibiting an agr response. In another embodiment of the present invention, Z has a side chain comprising oxygen or nitrogen. In another embodiment of the present invention, Z provides a functional group that can be cyclized through a thioether, ether or carbon-carbon group. In yet another embodiment of the present invention, the cyclic bond is a lactam or lactone bond. In yet another embodiment, y is 4. According to yet another embodiment of the present invention,
The peptide of the present invention comprises GVNAXSSLF (SEQ ID NO: 1),
G-A-N-A-X-S-S-S-L-F (SEQ ID NO: 2), G-V-A-A-X-
SSLF (SEQ ID NO: 3), AVANAXSSLF (SEQ ID NO: 4), GV-N-A-X-A- SLF (SEQ ID NO: 5), GV-
N-A-X-S-A-L-F (SEQ ID NO: 6), GV-N-A-A-X-S-S-S
It has an amino acid sequence containing AF (SEQ ID NO: 7) or XSSLF (SEQ ID NO: 8). Further, in yet another embodiment of the invention, the peptide of the invention has an amino acid sequence comprising a hydrophobic amino acid at the carboxy-terminal or pre-carboxy terminal position.

The present invention also provides a pharmaceutical composition comprising the desired peptide and a pharmaceutically acceptable carrier. In one embodiment of the invention, the carrier is selected from the group consisting of a diluent, an aerosol, a topical carrier, an aqueous solution, a non-aqueous solution and a solid carrier.

Finally, the present invention relates to Administering to the subject an effective amount of a pharmaceutical composition provided to treat the S. aures infection. Provided are methods for treating S. aures infection.

As used herein, “pharmaceutically acceptable” refers to molecular components that are physiologically acceptable and generally do not produce an allergic or similar deleterious reaction such as increased gastric abnormalities, dizziness, and the like. Refers to a composition. Pharmaceutically acceptable carriers include aqueous pharmaceutical solutions such as phosphate-interfered saline, oil / water emulsions or triglyceride emulsions, various types of wetting agents, standard pharmaceuticals such as tablets, coated tablets and capsules. And any of the acceptable carriers. Typically, such carriers contain excipients such as starch, milk, sugar, certain clays, gelatin, stearic acid, talc, vegetable oils or oils, gums, glycols or other well-known excipients. I do. Such carriers may include flavor and color additives or other ingredients. The present invention also provides suitable diluents, preservatives, solubilizers,
S.A. with emulsifiers and adjuvants. Provided is a pharmaceutical composition capable of inhibiting S. aures infection. Other embodiments of the compositions of the present invention include various forms of administration including, but not limited to, granular forms, protective coatings, protease inhibitors or intravenous, intramuscular, parenteral, pulmonary, nasal, and oral. Incorporate a permeation enhancer for

As used herein, an “effective amount” refers to the S.D. Aureus (S. aures)
) An amount necessary to reduce the infection by a clinically effective amount, preferably at least 30 percent, more preferably at least 50 percent, and most preferably at least 90 percent. Thus, the effective amount will depend on the subject being treated and the condition being treated. For purposes of the present invention, the method of administration should include, but is not limited to, transdermal, intradermal, intravenous, parenteral, oral, topical or aerosol administration.

The present invention further contemplates therapeutic compositions useful in practicing the treatment methods of the present invention. Therapeutic compositions of the present invention are described herein as active ingredients, as a mixture, of pharmaceutically acceptable excipients (carriers) and provided peptide or peptide compositions, compositions of peptidomimetics. One or more polypeptide analogs or fragments. Mixtures of various combinations of the provided pharmaceutical compositions are also contemplated.

The formulation of therapeutic compositions containing a polypeptide, analog or active fragment as an active ingredient is well understood in the art. Typically, such compositions are formulated as injectables, such as solutions or suspensions, but solid forms suitable for solution or suspension upon injection may also be formulated. it can. The formulation may be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline,
Dextrose, glycerol, ethanol, and the like, and combinations thereof. If desired, the compositions may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, which enhance the effectiveness of the active ingredient.

The polypeptide, analog or active fragment can be formulated as a therapeutic composition in the form of a pharmaceutically acceptable neutralized salt. Pharmaceutically acceptable salts include, for example, inorganic acids such as hydrochloric acid or phosphoric acid or acetic acid, oxalic acid,
Includes acid addition salts formed with organic acids such as tartaric acid, mandelic acid and the like (formed with free amino groups of polypeptide or antibody molecules). Salts formed from free carboxyl groups include, for example, inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide and isopropylamine, trimethylamine,
-It can also be derived from organic bases such as ethylaminoethanol, histidine, procaine and the like.

Therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, for example, by injection of a unit dose. The term `` unit dose '' when used in reference to the therapeutic composition of the present invention is suitable as a unit dose for humans, where each unit is composed of the required diluent, i.e. carrier or base. A physically discrete packaged unit containing a predetermined amount of active substance calculated to produce the desired desired therapeutic effect.

The compositions of the present invention can be prepared in a manner comparable to the dosage form
A therapeutically effective amount is administered. The quantity to be administered depends on the subject to be treated, the ability of the subject's immune system to utilize the active ingredient, and the degree of inhibition desired. Precise amounts of active ingredient required to be administered depend on the judgment of the attending physician and are peculiar to each individual. But,
Suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, more preferably 1 to several milligrams of active ingredient per kg of individual body weight per day. , Depending on the route of administration. Suitable treatment regimes for initial administration and booster administration can also be varied, but are typically followed by initial administration followed by repeated injections or other administration methods at intervals of one or more hours or more. You. Alternatively, continuous intravenous administration sufficient to maintain a blood concentration of 10 nanomolar to 10 micromolar is contemplated.

As used herein, “pM” means picomolar, “nM” means nanomolar, “uM” means micromolar, “mM” means millimolar,
“Ul” or “μl” means microliter, “ml” means milliliter, and “l” means liter.

As used herein, the term “synthetic amino acid” refers to an amino acid that is chemically synthesized and is not one of the 20 naturally occurring amino acids in nature.

As used herein, the term “biosynthetic amino acid” refers to amino acids found in nature other than the 20 amino acids commonly described and understood in the art as “natural amino acids” Means Examples of "non-amide isotopes" include secondary amine, ketone, carbon-carbon, thioether and ether moieties,
Not limited to them.

As used herein, the term “unnatural peptide analog” refers to a peptide variant that contains synthetic amino acids.

As used herein, amino acid residues are preferably in the “L” isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, so long as the desired functional property of binding to immunoglobulin is preserved by the polypeptide. NH ₂ refers to a free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of the polypeptide. Abbreviations for amino acid residues are described in J. Am. Biol. Chem.
, 243: 3552-59 (1969), in accordance with standard polypeptide nomenclature.

It should be noted that the sequence of all amino acid residues is represented herein by a formula in which the left and right orientations are the conventional directions from the amino terminus to the carboxy terminus. In addition, the dash at the beginning or end of the amino acid residue sequence
It should be noted that one or more amino acid residues exhibit a peptide bond to yet another sequence.

[0041] Amino acids having a non-polar R group include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine. Amino acids with unloaded electrodeic R groups include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Charged electrode R group (P
Amino acids having a negative charge at h6.0) include aspartic acid and glutamic acid. Basic amino acids (positively charged at pH 6.0) include lysine, arginine and histidine (at pH 6.0). Amino acids having a phenyl group include phenylalanine, tryptophan and tyrosine. Particularly preferred substitutions are the substitution of Arg and Lys and vice versa, where the positive charge is maintained, the substitution of Asp and Glu and vice versa, the negative charge is maintained and the substitution of Thr and Ser. To maintain the free -OH, Asn and Gl
Substitution of n while maintaining free NH ₂ . Amino acids are "D" or "L"
It may be an arrangement. The use of peptidomimetics involves the introduction of a non-amino acid residue having a non-amide bond at a predetermined position.

[0042] Amino acid substitutions can also be introduced to substitute amino acids having particularly favorable properties. For example, a Cys can introduce a site capable of disulfide bonding with another Cys. His can be introduced specifically as a “catalytic” site (ie, His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced due to a particularly flat structure that induces a β-turn in the structure of the protein. The following examples are provided to more fully illustrate the preferred embodiments of the present invention. However, they should not be considered as limiting the scope of the invention. Although the present invention is illustrated herein with reference to various specific materials, procedures and examples, the present invention is limited to the specific materials, combinations of materials and procedures selected for that purpose. It is understood that it will not be. As will be appreciated by those skilled in the art,
Numerous changes in such details can be included.

[0043]

【Example】

Example 1 Synthesis of a novel class of peptides responsible for S. aures bacterial interference

The first synthetic route to AgrD thiololactone peptides, including the solvent-based cyclization of thiol ester precursors, has proven problematic due to the difficulties involved in synthesizing linear starting materials. I have. Therefore, a more efficient method was developed that included solid-phase cyclization as the last synthetic step (FIG. 1A). A key feature of this method was the ability to produce fully deprotected peptides immobilized on a solid support via a reactive thiol ester linkage. This is the recently described 3-mercaptopropionamide-polyethylene glycol-poly (N, N-dimethylacrylamide) [
HS-PEGA] support (Camarero, JA, 1998)
To Boc-Solid Phase Peptide Synthesis (SPPS) (Schnolzer)
, M) et al., 1992). The acid stability of the alkyl-thiol ester bond between the peptide and the resin means that the completed peptide can be totally deprotected without being cleaved from the support. The formation of the desired thiololactone-peptide is then performed simply by swelling the peptide-resin beads in a buffered aqueous solution at pH 7.0, resulting in a chemoselective intramolecular cyclization / cleavage reaction. This last step is
It should be noted that this is possible due to the excellent swelling properties of the PEGA support in water (Mendal, M., 1992).

The synthesizing method illustrated in FIG. Aureus (S. aures)
It was used to produce Group I and Group II strains of the peptides AgrDI and AgrDII (the amino acid sequence of the Group III Agr peptide was not established at the time of the present invention). In each case, the last cyclization / cleavage reaction was remarkably complete, yielding one major component in excellent yield (FIG. 1B). After purification, the coupled product is
Electrospray mass spectrometry, chemical reactivity to neutral hydroxylamine (Chou, TC and Lipmann, F.), 1
952) and two-dimensional ¹ H nuclear magnetic resonance (NMR) spectroscopy (Bax, A
. ) Et al., 1985; Wuthrich, K., 1986). (The peptide thiol ester reacts quickly with neutral hydroxylamine to yield the corresponding hydroxamate derivative).

The biological activity of the synthetic AgrDI and AgrDII peptides was determined by the culture S. aureus containing a β-lactamase reporter gene fused to the agrP3 promoter. Assays were performed using S. aures strains (Novic
k, R. P) et al., 1995). Thus, activation or inhibition of the agr response can be monitored spectrophotometrically using a chromogenic β-lactamase activity assay (Table 1). As with its naturally derived counterpart, synthetic Agr
DI and AgrDII have their own S.D. Activates the agr response only within the S. aures class and the other two classes of S. aures. Aureus (S.
aures) strain was found to inhibit the agr response only (Table 1). Further studies performed revealed a dose-dependent relationship between the amount of peptide added to the culture supernatant and the degree of activation / inhibition of the agr response (FIGS. 2A-2B). Analysis of the resulting sigmoid response curves indicated that the ED50 and IC50 values that activate and inhibit the agr response, respectively, were in the lower namomol range (Table 1).
. In addition, these data indicate that there is a critical threshold concentration of AgrD peptide required for activation of the agr response. This is consistent with the previously proposed density sensitivity / self-induction mechanism (Ji, G. et al., 1997). Importantly, no activation / inhibitory effect was detected with the linear carboxylate synthetic peptides corresponding to the AgrDI and AgrDII sequences, even at high μM concentrations (Table 1). Its biological activity is limited to thiololactone-peptides and serves to confirm that this unusual post-translational modification is present in the secreted AgrD peptide.

Example 2: Functional Importance of Ring Structures

The accuracy and simplicity of the synthetic methods of the present invention allow for the systematic alteration of the peptide's chemical structure, thereby allowing detailed structure-activity studies to be performed. For the AgrD peptide, the initial focus was on the following questions: (
i) Which amino acids in the sequence are critical for affinity / selectivity? (Ii)
What is the role of the thiololactone unit in activating and inhibiting the agr response? To address the first of these issues, the group II Agr
An alanine scan was performed on the D peptide. Each of the alanine-modified AgrDII peptide variants was produced and characterized as described above. In each case, the purified peptide had three S. cerevisiae species Each of the S. aures strains was assayed for its ability to activate or inhibit the agr response. The analysis of the results is summarized in FIG.
It has been shown that certain amino acids remain both within the ring and at the end of the molecule important for activation of the agr response (Asn-3, Leu-8, Phe-9). Alanine peptide mutants exhibit a broad activation profile, exhibiting both increased activity and phenomena. On the other hand, the inhibition of the agr response seems to depend on only the amino acids in the ring (Leu-8, Phe-9), and the alanine-modified AgrDI exhibiting the inhibitory action.
All I peptides show higher activity compared to wild type.

The functional importance of the thiololactone structure of the AgrD peptide was investigated by the synthesis of a series of AgrDII variants (Table 1). To address whether a cyclic thiol ester group is strictly required for the activity of the native peptide, a linear thiol ester analog of AgrDII was prepared and assayed (see experimental protocol for details). ). As in the case of the linear carboxylate AgrDII variant described above, the linear thiol ester peptide failed to activate or inhibit the agr response even when μM concentrations were added to the cultured cells. This result suggests that the cyclic structure present in the AgrD peptide is essential for biological activity.

Example 3 Cyclic Lactone and Lactam Variants Provide a Peptide That Enables Inhibitory Action Without Activating Action

The thiol ester group is a moderately good acylating agent and has been used in several biological processes (Law, SK) and Dodge (AW Dodds).
Xu, M.-Q. and Parlor (FB Perler), 1997;
Porter, JA, et al., 1996). It is interesting to speculate that upon acceptor binding, the thiololactone present in the AgrD peptide acts as an acyl donor for covalent modification of particular residues in AgrC. Thiololactone unit in AgrDII is converted to ester (lactone)
The effect of replacing with amide moieties (lactams) was interesting. In principle, both of these variants should be significantly less reactive than the thioester peptide (), but the lactone variant of AgrDII should also be a wild-type isostere. The synthesis of the desired AgrDII lactone and lactam variants was performed by solution cyclization of the partially protected intermediate, followed by overall side chain deprotection. In particular, thiol esters were significantly more reactive with nitrogen nucleophiles than oxygen esters (Bruice, TC) and Benkovic.
ic), 1966). As in the case of the chain peptides described above, the purified lactone and lactam variants are three S. aureus. No agr response could be activated in any of the S. aures strains (Table 1). However, both variants are I
S. group and III group S. It can inhibit the agr response of S. aures strains. Clearly, the reactive thioester linkage is agr in vivo.
Required for activation of the response, but not for inhibition.

Analysis of the biological properties of the various AgrDII peptide variants produced in this study reveals that: (i) activation of the agr response depends on the amino acid sequence and chemical / stereochemical properties of the AgrD peptide. Very sensitive to both, (ii
A) Inhibition of the Agr response is sensitive to the backbone stereochemistry and amino acid sequence of the AgrD peptide, but is not affected by changes in the chemical reactivity of the cyclic bonds. These observations suggest that activation and inhibition of the agr response occurs by two different mechanisms.

The synthesis of Staphylococcus aureus virulence factors and other extracellular proteins responsible for virulence is under the control of the agr locus.
The peptide AgrD encoded by the secreted agr is known to be an effector of self-activation of the agr response and cross-strain inhibition. Preliminary analysis of AgrD peptides isolated from culture supernatants suggested that they contained an unusual thiol ester linked cyclic structure. In the present invention, the chemical synthesis comprises:
These AgrD peptides confirm that they contain a thiololactone unit and that this structure is absolutely necessary for the full biological activity of the native peptide.

The structure-function studies provided by the present invention identify and elucidate key aspects of the peptide structure involved in the differential activation and inhibitory functions of the peptide. Also provided are novel, non-naturally occurring peptide variants that exhibit no activating effect and retain (or enhance) the inhibitory effect. Peptides having such properties are described in Aureus (S. au
res) Useful for treating infections.

[0055]

[Discussion]

The AgrD peptide has its own A in a different manner than another class of AgrC receptors.
One proposed model that binds to the grC receptor (ie, the same S. aureus class) is illustrated in FIG. This involves two slightly different orientations of the peptide within the receptor binding pocket, but cannot exclude two or more distinct binding sites within the AgrC receptor. Intra-class relationships further assume that the highly specific side chain interaction between AgrC and AgrD places the thiol ester bond of the peptide adjacent to a nucleophilic group in the receptor. Such close proximity results in the activation of the transacylation reaction and the agr response by the associated conformational change of the receptor. The thiol ester linkage is a moiety that is reactive enough to participate in this trans-acylation step, and is one of several conventional methods that result in stereochemical inhibition such as disulfide- or amide-bond formation. But describes its presence within the AgrD peptide.

On the other hand, the absence of these specific interactions has led to the
It does not appear to be so close in ligand interaction. Thus, inhibition of the agr response is not involved in the signaling transacylation step, but rather in non-covalent interactions that may serve to eliminate the strain's own self-induced AgrD peptide from the receptor binding pocket. I do. This non-covalent interaction requires specific residues, as shown by the results of the alanine scan mutation. It is appropriate to note that lactone and lactam variants can be inhibited in an appropriate interclass manner, while not inhibiting themselves. Therefore,
Inhibition of initialization does not occur if the strain's own self-inducible lactone / lactam variant encounters its own receptor. This evidence further supports the proposed model that activation and inhibition occur by distinct mechanisms.

In summary, chemical synthesis was used to confirm the presence of a thiololactone structure within the secreted AgrD self-inducing peptide. This extremely rare post-translational modification appears to be essential for full biological activity. Based on initial structure-activity studies, the thiol ester functionality undergoes a transacylation reaction by the specific nucleophile of the AgrC receptor, and this chemical step is ag
It is discussed that it is required for activation of the r response (but not for inhibition). Thiololactone units also limit the in vivo half-life of the peptide, and thiol ester linkages can undergo slow hydrolysis at physiological pH to form inactive linear peptides. The biological significance of this short-term in vivo effect is still unknown. The 38 kDa RAP protein currently reported (Balaban (
Balaban, N) et al., 1998) Ag by secreted AgrD peptide.
It is also unclear what role, if any, plays in activating the r response.

Access to the AgrD peptide by facile synthesis is a key step in using the agr self-inducing system as a route to novel therapeutic agents. Indeed, the observation that a novel synthetic AgrD variant can be produced that can inhibit the agr response but not activate it is particularly important in this regard. further,
The ability to easily adapt solid phase synthesis methods to combinatorial synthesis is advantageous for rapid identification of interesting compounds.

[0059]

[Experiment procedure]

 Biological activity of the synthetic AgrD peptide: The bioassay was performed using agrP3-blaZ fusion
(Novick, RP, et al., 1995).
), Group I (RN6390B), Group II (SA502A) and Group III (RN8
463) S.M. S. aures strain (Ji, G., et al., 1997).
). Cells were incubated with agr activated in CYGP medium at 37 ° C.
Studies grew to early exponential phase and agr inhibition studies grew to mid exponential phase. Then
Cultured cells were treated with buffer (negative control) as described (Ji, G. et al., 1995).
A suitable cell suspension containing the produced native AgrD peptide (positive control) or
The synthetic peptide was prepared using tris. Solution added to 20 mM HCl buffer
Some of them were added. The culture is then shaken at 37 ° C. for 55 minutes (
Incubation (activation) or 80 minutes (inhibition), (Novik
ck, R.A. P. ) Et al., 1995).
Β-lactamase activity was assayed using fluorescence spectroscopy. ED₅₀And IC ₅₀ Is the program PRISM (GRAPHPAD Software Inc. (GR
APHPAD Software Inc,), San Diego, CA
) Was used to extract a sigmoidal dose-response curve (eg, FIGS. 2A-2B).
. All assays were performed in triplicate and ED₅₀And IC₅₀Value is within ± 10%
Was in

[0060]

[Research on thioesterification]

Initial work was performed on a chain α using 5,5′-dithiobis (2-nitrobenzoic acid).
-For selective thioesterification of thiolic acid peptides, S- (5-sulfenyl-2-nitrobenzoic acid) α-thiol ester derivatives were obtained. This linear thiol ester precursor was then cyclized in solution by transthioesterification involving the sulfhydryl group of cysteine. The overall yield using this method is:
It was low mainly due to the difficulty in selectively esterifying the α-thiol peptide.

[0061]

[Peptide synthesis]

All peptides were synthesized manually by the Boc SPPS in situ neutralization / HBTU activation protocol (M. Schnolzer).
), Allewood (P. Alewood), Jones (A. Jones), Allewood (D. Alewood), Kent (BH Kent), Int. J. P
ept. Protein Res. 40: 180-193 (1992). Boc-AA- [COS] -P to which thiololactone AgrD peptide has been added in advance
Assembled on EGA (Camarero, JA, et al., 1998).
. After chain assembly, the peptide was treated with HF at 0 ° C. for 1 hour to yield the corresponding fully deprotected peptide- [COS] -PEGA resin, then chilled diethyl ether, then 0.1% trifluoro CH ₃ CN / H ₂ O containing acetic acid
And washed. The deprotected peptide is cyclized chemoselectively to a pH of 0.1 at pH 7.0.
The beads were simultaneously cut from the support by swelling the beads in a mixture of M sodium phosphate buffer and acetonitrile (80:20). After the reaction for 12 hours, the beads were filtered off, washed with a 0.1% aqueous trifluoroacetic acid solution, and the peptide was subjected to reverse phase HP.
Purified from the filtrate by LC.

[0062]

[Assembling peptides on resin]

Peptide GVNAASSLF was purified using HS-PEGA using Boc-SPPS.
Resins (Camarero et al., 1998) were assembled. This is 1
Two cysteine residues correspond to the AgrDII sequence mutated to alanine. After synthesis and overall deprotection, the peptide- [COS] -PEGA beads were swollen in a buffer containing 0.1 M sodium phosphate, pH 7.0 and ethanethiol (2% v / v) and the cleavage reaction was performed. Proceeded for 3 hours. The desired ethyl α-thiol ester peptide was then purified from the supernatant by reverse phase HPLC.

[0063]

[Protected peptide]

Cys5 is mutated to Ser (lactone) and Cys5 is Dapa (
Protected peptide Z-Gly-Val-Asn-Ala-Ser ('Bu) -S corresponding to the AgrDII sequence mutated (lactam)
er (Bzl) -Ser (Bzl) -Leu-Phe and Z-Gly-Val
-Asn-Ala-Dapa (Boc) -Ser (Bzl) -Ser (Bzl)
-Leu-Phe was synthesized on Wang-resin using the Fmoc Nα protection method using the HBTU activation protocol. After chain assembly, the peptide was cleaved from the support and the Ser-5 or Dapa-5 side chains were deprotected by treatment with a mixture of trifluoroacetic acid: anisole: water (90: 5: 5) for 4 hours. The partially protected peptide-α carboxylate was then converted to DMF (0.5 mg / m
L) and treated with PyBOP (5 equivalents) (and the lactone precursor was a catalytic amount of dimethylaminopyridine). The cyclization reaction was monitored by HPLC and the complete reaction took 2 hours. The remaining protecting groups were then removed by treatment with HF, and the desired peptide was purified by reverse phase HPLC and analyzed by mass spectrometry and 2D ¹ HN.
Characterized by MR spectrometer.

[0064]

[NMR]

Two-dimensional ¹ H NMR spectra were measured on a Bruker DPX-400 spectrometer. The peptide was dimethyl sulfoxide-d6 or 9: 1 H2O / D2O pH4
. 0 to a final concentration of 1-2 mM and a series of TOCSY (Bax (AB)
ax), DG Davis, J. et al. Magn. Reson. 65:
355-360 (1985) and ROESY experiments were recorded at 278K. ¹ H
The resonance was transferred by the usual procedure (K. Wuthrich, NMR of
Proteins and Nucleic Acids (Wiley,
New York, 1986).

[0065]

[References]

The following is a list of documents relating to the above disclosure, especially experimental procedures and considerations.
The document should be considered as being incorporated by reference in its entirety. 1. Balaban, N., et al., Science 280: 438-440 (1998). 2. Bax, A. and DG Davis, J. Magn. Reson. 655: 355-360 (1985). 3. Buice, TC and SJ Benkovic, in Bioorganic Mechanisms 1: 259-297 (Ben
jamin, New York, 1966). 4. Camarero, GC, et al ,, J. Peptide Res. 51: 303-316 (1998) .5.Chou, TC and F. Lippmann, J. Biol. Chem, 196: 89 (1952). 6. Ji, G., et al., Proc. Natl. Acad. Sci. USA 92: 12055-12059 (1995) .7. Ji, G., et al., Science 276: 2027-. 2030 (1997). 8. Law, SK, and AW Dodds, Protein Science 6: 263-274 (1997) .9.Mendal, M. Tetrahedron Lett. , et al., EMBO J. 14: 4569-4577 (1995) .11. Muir, TW, et al., Chemistry and Biology 3: 817-825 (1996) .12.Novick, RP, et al., Mol. Gen. Genet. 248: 446-458 (1995). 13. Novick, RP, et al., EMBO J. 12: 3967-3975 (1993). 14. Porter, JA, Science 274: 255-259 (1996). 15. Schnolzer, M .., et al., Int. J. Pept. Protein Res. 40: 180-193 (1992)
16. Wulthrich, K. NMR of Proteins and Nucleic Acids (Wiley, New York, 19
86). 17.Xu, M.-Q. and FB Perler, EMBO J. 15: 5146-5133 (1996).

The present invention is capable of other embodiments and of being practiced in other ways without departing from the spirit or essential characteristics of the invention. Accordingly, the disclosure of the present invention is to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims and falling within the meaning and range of equivalency. All modifications are intended to be included in the present invention.

[Sequence list]

[Brief description of the drawings]

FIG. 1A is a chemical structure of an AgrD self-inducing peptide, showing formation of a thiololactone peptide by solid-phase intramolecular chemical bonding.

FIG. 1B is the chemical structure of an AgrD self-inducing peptide, reverse phase HPLC of a crude reaction mixture of AgrDI peptide synthesis. The inset shows the electrospray mass spectroscopy (ESMS) obtained from the major components in the mixture: expected mass of AgrDI thiololactone = 962.374 Da (961.9 mass).

FIG. 1C is the reversed-phase HPLC of the crude AgrDII reaction mixture with the chemical structure of the AgrD self-inducing peptide. The inset shows the ESMS of the major components in the mixture: AgrDII
Expected mass of thiololactone = 880.129 Da (879.9 mass). Both HPLC spectra were obtained using a 30 minute linear gradient of 0-73% buffer B (buffer B = CH ₃ CN: H ₂ O: trifluoroacetic acid; 90: 10: 1).
). ESMS was performed on a PE-Sciex API-100 single quadrupole electrospray mass spectrometer. The calculated value of mass is calculated by the program MacProMas.
s (Sunil Vemuri and Terry Lee)
nd Terry Lee), City of Hope (City of Hope)
e), Dualte, CA).

FIG. 2A shows representative data for activation and inhibition of the agr response by synthetic thiololactone peptides, respectively, where the synthetic thiololactone peptide is biologically active.
The extent of activation / inhibition of the agr response based on β-lactamase activity (see Table 1) is shown as a plot of Vmax versus peptide concentration. Group II S. Aureus (S.
aures) Activation of agr response by synthetic AgrDII in cells.

FIG. 2B shows representative data for activation and inhibition of the agr response by synthetic thiololactone peptides, respectively, where the synthetic thiololactone peptide is biologically active.
The extent of activation / inhibition of the agr response based on β-lactamase activity (see Table 1) is shown as a plot of Vmax versus peptide concentration. Group I S. Aureus (Sa
(ures) Inhibition of agr response in cells by synthetic AgrII.

FIG. 2C shows representative data for activation and inhibition of the agr response by synthetic thiololactone peptides, respectively, where the synthetic thiololactone peptide is biologically active.
The extent of activation / inhibition of the agr response based on β-lactamase activity (see Table 1) is shown as a plot of Vmax versus peptide concentration. FIG. 7 shows the effect of replacing each residue in the AgrDII sequence with alanine on activation and inhibition.

FIG. 3A shows a proposed model of activation and inhibition of the agr response. Activation of the agr response indicates that self-AgrD peptides occur via intra-class interactions that interact with self-AgrC receptors. The specific AgrD / AgrC interaction causes the peptide to be properly positioned to undergo acyl transfer by a nucleophile in the receptor, resulting in a signaling conformational change.

FIG. 3B shows a proposed model of activation and inhibition of the agr response. Inhibition of the agr response is shown to occur by non-covalent interactions between classes that act to eliminate the strain's own activating peptide from the receptor. This interaction is also specific.

[Procedure amendment]

[Submission date] February 8, 2001 (2001.2.8)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG. 1A

FIG. 1B

FIG. 1C

FIG. 2A

FIG. 2B

FIG. 2C

FIG. 3A

FIG. 3B

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Mayville Patricia United States of America, New York 10021, York Avenue, New York # 132 1230 (72) Inventor Novic Richard P. United States, New York 10024, New York, West 86 Street 10 (72) Inventor J.A. B 211 F F term (reference) 4C084 AA02 BA25 BA26 DA43 MA13 MA17 MA34 NA14 ZB351 ZB352 4H045 AA10 BA11 BA12 BA13 BA14 BA15 BA16 BA17 BA31 CA11 EA29 FA53 FA57 FA61 GA45

Claims (24)

[Claims] 1. The structure, Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres, Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, and R is oxygen, nitrogen and carbon Is selected from the group consisting of
And y is 1-10). 2. A cyclic peptide containing an amino acid sequence of NH ₂ -X _(n) -ZX _(y) -COOH and a cyclic bond other than a thioester between Z residue and COOH (where X is an amino acid, Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, n is 0-10, and y is 1-10, selected from the group consisting of amino acid analogs, peptidomimetics and non-amide isosteres ). 3. Z has a side chain containing oxygen, nitrogen or carbon.
3. The cyclic peptide according to [1]. 4. The cyclic peptide according to claim 2, wherein said cyclic bond is a lactam or lactone bond. 5. The cyclic peptide according to claim 1, which is capable of inhibiting an agr response. 6. The cyclic peptide according to claim 2, which is capable of inhibiting an agr response. 7. The cyclic peptide according to claim 1, wherein y is 4. 8. The cyclic peptide according to claim 2, wherein y is 4. 9. GV-N-A-A-X-S-S-S-L-F (SEQ ID NO: 1), G
-A-N-A-X-S-S-S-L-F (SEQ ID NO: 2), G-V-A-A-X-S
-SLF (SEQ ID NO: 3), AVANAX-XSSLF (SEQ ID NO: 4), GVN-A-X-A-S-S -LF (SEQ ID NO: 5), GVN
-A-X-S-A-L-F (SEQ ID NO: 6), GV-N-A-X-S-S-A
The cyclic peptide according to claim 7, which has an amino acid sequence containing -F (SEQ ID NO: 7) or XSSLF (SEQ ID NO: 8). 10. GV-N-A-A-X-S-S-S-L-F (SEQ ID NO: 1),
G-A-N-A-X-S-S-S-L-F (SEQ ID NO: 2), G-V-A-A-X-
SSLF (SEQ ID NO: 3), AVANAXSSLF (SEQ ID NO: 4), GV-N-A-X-A- SLF (SEQ ID NO: 5), GV-
N-A-X-S-A-L-F (SEQ ID NO: 6), GV-N-A-A-X-S-S-S
The cyclic peptide according to claim 8, which has an amino acid sequence comprising AF (SEQ ID NO: 7) or XSSLF (SEQ ID NO: 8). 11. A composition comprising the peptide according to claim 1 and a carrier. 12. A composition comprising the peptide according to claim 2 and a carrier. 13. A pharmaceutical composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier. 14. A pharmaceutical composition comprising the peptide of claim 2 and a pharmaceutically acceptable carrier. 15. The method according to claim 15, wherein the carrier is a diluent, an aerosol, a topical carrier, an aqueous solution,
14. The pharmaceutical composition according to claim 13, wherein the composition is selected from the group consisting of a non-aqueous solution and a solid carrier. 16. The method according to claim 16, wherein the carrier is a diluent, an aerosol, a topical carrier, an aqueous solution,
15. The pharmaceutical composition according to claim 14, wherein the composition is selected from the group consisting of a non-aqueous solution and a solid carrier. 17. The S.T. 16. A method for treating an S. aures infection, comprising administering to a subject an effective amount of the pharmaceutical composition of claim 15 to treat the infection. 18. The S.I. 17. A method for treating an S. aures infection, comprising administering to a subject an effective amount of the pharmaceutical composition of claim 16 to treat the infection. 19. The structure, Wherein X is selected from the group consisting of amino acids, amino acid analogs, peptidomimetics and non-amide isosteres, Z is selected from the group consisting of synthetic amino acids and biosynthetic amino acids, and R is oxygen, nitrogen and carbon Is selected from the group consisting of
And y is from 1 to 10). A. assembling the linear peptide component being manufactured on the PEGA resin support to form a protected, attached peptide chain; B. treating and deprotecting the peptide chain of step A; B. cleaving the deprotected peptide of step B with a buffer at neutral pH from the solid support and treating the peptide for a period of time sufficient to form the cyclic peptide; Recovering the cyclic peptide. 20. A method for producing a cyclic peptide according to claim 1, comprising: A linear peptide chain having a composition corresponding to the cyclic peptide is applied to a solid resin support containing 3-mercapto-propionamide-polyethylene glycol-poly- (N ₁ N ₄ -dimethacrylamide) (HS-PEGA). B. assembling to form a protected peptide assembly; B. treating the protected peptide assembly of step A to deprotect said peptide assembly; Treating the deprotected peptide of Step B with an aqueous buffer solution at a pH of about 7.0 for a time sufficient to form the cyclic peptide and cleave the peptide from the solid phase resin support; D . Recovering the cyclic peptide. 21. The method of claim 19, wherein said solid phase resin support comprises BOC-AA- (chain peptide assembly) -PEGA. 22. The method of claim 19, wherein the processing of step B is performed using HF for about 1 hour. 23. The process of step C, wherein the process of step C is performed using a buffer comprising Na ₂ PO ₄ and acetonitride.
9. The method according to 9. 24. The method of claim 22, wherein the processing of Step C is performed for about 12 hours.