Proteas stabilized antibacterial peptides for S. aureus

Abstract

Improved peptide compositions and synbody compositions are disclosed that show improved stability and antibiotic activity. The new antibacterial peptides for S. aureus have particular D-amino acid substitutions in order to increase protease stability while also preserving marked antibiotic activity. Thus, compositions and methods for treating infections related to S. aureus also are disclosed.

Claims

1. A bactericidal composition comprising a peptide scaffold ScO: ##STR00001## conjugated to at least one other peptide having at least one D-amino acid.

2. The composition of claim 1, wherein said composition comprises synbody Ly-Ly-ScO: ##STR00002## wherein lowercase letters represent D amino acids.

3. The composition of claim 1, wherein said composition is selected from the group consisting of synbody D1-D1-ScO (CSGkkRRHHrrkkRrHHrrK-ScO-CSGkkRRHHrrkkRrHHrrK), synbody D2-D2-ScO (CSGRrkrPrrkrPRrkRPRr-ScO-CSGRrkrPrrkrPRrkRPRr), and synbody D2-88-ScO (CSGRrkrPrrkrPRrkRPRr-ScO-CSGEMWAIMPPIIKPDNKGH), wherein lowercase letters represent D amino acids.

4. A method of treating an S. aureus infection, comprising the step of administering an effective amount of synbody Ly-Ly-ScO: ##STR00003## to an infected subject, wherein lowercase letters represent D amino acids.

5. A method of treating an S. aureus infection, comprising the step of administering an effective amount of synbody D1-D1-ScO, (CSGkkRRHHrrkkRrHHrrK -ScO-CSGkkRRHHrrkkRrHHrrK), synbody D2-D2-ScO (CSGRrkrPrrkrPRrkRPRr-ScO-CSGRrkrPrrkrPRrkRPRr), and synbody D2-88-ScO (CSGRrkrPrrkrPRrkRPRr -ScO-CSGEMWAIMPPIIKPDNKGH), wherein lowercase letters represent D amino acids, to an infected subject, and wherein ScO comprises: ##STR00004##

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 depicts a D-amino acid substituted synbody, termed synbody (4).

(2) FIG. 2 depicts a bivalent scaffold for synbody construction (ScO).

(3) FIG. 3 depicts an amino acid sequence of peptide Ly (depicted as the R group) and a D-amino acid substituted bivalent peptide (Ly-Ly-ScO).

(4) FIG. 4 shows the bactericidal activity of Ly-Ly-ScO after 14 hour incubation with either 10.sup.7 CFU/mL (open circles) or 10.sup.9 CFU/mL (black squares) MSSA culture as a function of Ly-Ly-ScO concentration.

DETAILED DESCRIPTION

(5) Novel peptides and bivalent peptides (synbodies) have been produced that include selective substitution of L-Arg and L-Lys with D-Arg and D-Lys, in conjugation with N-terminal acylation. Exemplary synbodies (FIG. 1 and FIG. 3B) that are more protease stable and active against both MSSA and MRSA (Table 1 and Table 2) are further described below.

(6) TABLE-US-00001 TABLE 1 Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC), half-life in serum, and hemolysis from synbody (4). Three different strains of MSSA and 2 different strains of MRSA were tested. MSSA MRSA (n = 3) (n = 2) H.sub.50 t.sub.1/2 Synbody (4) 2.2 μM 3.3 μM >200 μM ~130 min H.sub.50 = concentration that causes 50% hemolysis t.sub.1/2 = in vitro half-life in mouse serum

(7) While synbody (4) is protease stable and had some bactericidal activity against MSSA, the molecule is synthesized by solid phase peptide synthesis with a low final yield. This increases the final cost of the molecule which has historically been one of the major impediments to widespread use of peptide antibiotics. To overcome this limitation, we have employed a conjugation strategy in which the purified peptide arms are conjugated to the bivalent peptide scaffold (ScO, FIG. 2) through maleimide chemistry.

(8) Conjugation to the scaffold occurs through the thiol of a terminal Cys (either N-terminus or C-terminus) and the reaction proceeds to completion after ˜12 hours incubation at room temperature. The final synbody is then purified by HPLC and the mass of the synbody is confirmed by MALDI.

(9) We used this conjugation approach to conjugate the protease stabilized lytic peptide arm of synbody (4), called Ly, to ScO and produce a new anti-S. aureus peptide (Ly-Ly-ScO, FIG. 3). Lower case amino acids signify D-AA version of the amino acid. For example, r corresponds to D-Arg. To produce Ly-Ly-ScO, 2.2 molar equivalents of peptide (FIG. 3) were added to 1 equivalent of ScO in aqueous buffer (1×phosphate buffered saline) at pH 7. The reaction incubated at room temperature for 24 hours and was then purified using reverse phase HPLC with standard methods.

(10) TABLE-US-00002 TABLE 2 MIC, MBC, half-life in serum, and hemolysis from Ly-Ly-Sc0. MIC (μM) MRSA MBC (μM) MSSA (USA300) MSSA t.sub.1/2 H.sub.50 Ly-Ly-Sc0 12.5 6.25 12.5 not tested >500 uM

(11) Additionally, we have developed new peptides of mixed D/L AA composition that are conjugated using the ScO bivalent peptide scaffold (FIG. 2). These peptides have similar activities against MSSA and are also bactericidal, with low red blood cell toxicity. Thus, methods of treating S. aureus infections involve administering (by any known way) an effective amount of peptide or synbody described herein to an infected patient or subject.

(12) TABLE-US-00003 TABLE 3 MIC, Minimum bactericidal concentration (MBC), and hemolysis of D/L-AA composition peptides. MIC MBC (μM) (μM) H.sub.50 (MSSA) (MSSA) (μM) D1-D1- CSGkkRRHHrrkkRrHHrrK-Sc0-  10  5 >500 Sc0 CSGkkRRHHrrkkRrHHrrK D2-D2- CSGRrkrPrrkrPRrkRPRr-Sc0-   5  5 >500 Sc0 CSGRrkrPrrkrPRrkRPRr D2-88- CSGRrkrPrrkrPRrkRPRr-Sc0-  50 50 >500 SC0 CSGEMWAIMPPIIKPDNKGH D1 CSGkkRRHHrrkkRrHHrrK <50 n.t. D2 CSGRrkrPrrkrPRrkRPRr <50 n.t. 88 CSGEMWAIMPPIIKPDNKGH n.t. n.t. (SEQ ID NO. 1) n.t. = not tested. Lower case amino acids signify D-AA version of the amino acid. For example, r corresponds to D-Arg.

(13) It further is contemplated that the novel and inventive compositions described herein will be used in treatment methods not practiced on the human body. For example, animals may be treated.

(14) Various changes in the details and components that have been described may be made by those skilled in the art within the principles and scope of the invention herein described in the specification and defined in the appended claims. Therefore, while the content above has been shown and described herein in what is believed to be the most practical and preferred embodiments, it is recognized that departures can be made therefrom within the scope of the invention, which is not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent processes, compositions, and products.