# Patent

## Title
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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 62/029,948, filed Jul. 28, 2014, the entire contents of which are incorporated herein in their entirety by reference.


STATEMENT OF GOVERNMENT RIGHTS
This invention was made with government support under W911NF-10-1-0299 awarded by the Army Research Office. The government has certain rights in the invention.


TECHNICAL FIELD
This disclosure relates to the field of antibiotics and more particularly to bivalent peptides that possess antibiotic activity.
BACKGROUND
Antimicrobial resistance of bacteria is rapidly increasing and has been declared a multinational public health crisis. One of the most critical resistant pathogens is Methicillin Resistant Staphylococcus Aureus (MRSA), which has developed resistance to all beta-lactam antibiotics. There are 80,000 severe MRSA infections each year in the United States that are responsible for 11,285 deaths annually (CDC Antibiotic Resistance Threats in the United States, 2013).
While new antibiotics are in development, the ones closest to market are derivatives of old antibiotics. S. aureus has proven remarkably resilient and developed resistance to all introduced antibiotics, including a recent report of vancomycin resistant-MRSA. This suggests that resistance will rapidly develop against these new antibiotics as well.
SUMMARY
This disclosure relates to improved bivalent peptides that are useful as antibiotics. The first generation bivalent peptide, called a synbody, was bacteriostatic while improved versions were bactericidal with low toxicity. However, these synbodies are composed of L-amino acids (L-AA) and are inherently sensitive to protease degradation, both from endogenous proteases and from those secreted from S. aureus. 
To improve the protease stability of peptide therapeutics, selective D-amino acid (D-AA) substitution was used to produce new peptides that are resistant to degradation. However, often times the D-AA substituted antimicrobial peptides (AMP) are less active than the L-AA AMP.
Selective substitution of L-Arg and L-Lys with D-Arg and D-Lys, in conjugation with N-terminal Acylation, has been found to produce synbodies that are more protease stable and active against both MSSA and MRSA. Thus, improved peptide compositions and synbody compositions are disclosed herein that show improved stability and antibiotic activity.
Various other purposes and advantages will become clear from the description in the specification that follows. Therefore, this specification includes the features hereinafter fully described in the detailed description of the preferred embodiments, and particularly pointed out in the claims. However, such description discloses only some of the various embodiments and ways in which the invention may be practiced.



DESCRIPTION OF DRAWINGS
FIG. 1 depicts a D-amino acid substituted synbody, termed synbody (4).
FIG. 2 depicts a bivalent scaffold for synbody construction (ScO).
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).
FIG. 4 shows the bactericidal activity of Ly-Ly-ScO after 14 hour incubation with either 107 CFU/mL (open circles) or 109 CFU/mL (black squares) MSSA culture as a function of Ly-Ly-ScO concentration.



DETAILED DESCRIPTION
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.







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)
H50
t1/2

















Synbody (4)
2.2 μM
3.3 μM
>200 μM
~130 min







H50 = concentration that causes 50% hemolysis



t1/2 = in vitro half-life in mouse serum






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.
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.
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.







TABLE 2







MIC, MBC, half-life in serum, and hemolysis from Ly-Ly-Sc0.










MIC (μM)















MRSA
MBC (μM)





MSSA
(USA300)
MSSA
t1/2
H50
















Ly-Ly-Sc0
12.5
6.25
12.5
not tested
>500 uM









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.







TABLE 3







MIC, Minimum bactericidal concentration (MBC),


and hemolysis of D/L-AA composition peptides.













MIC
MBC





(μM)
(μM)
H50




(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.






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.
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.

## Claims

1. A bactericidal composition comprising a peptide scaffold ScO:




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:




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:




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: