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Title:
MULTIFUNCTIONAL ANTIBIOTIC-PEPTIDE CONJUGATES: SYNTHESIS AND ANTIMICROBIAL ACTIVITIES
Document Type and Number:
WIPO Patent Application WO/2018/085846
Kind Code:
A1
Abstract:
The invention relates to multi-functional antibiotic agents formed from antibiotics conjugated to microbial membrane disrupting peptides. These antibiotic-peptide conjugates are designed so that the conjugate simultaneously functions as a membrane disrupter and a metabolic inhibitor. In illustrative embodiments of the invention, the peptide of the conjugate causes the membrane of a bacteria to become permeable to the conjugate, and the antibiotic of the conjugate functions as a metabolic inhibitor by binding to bacterial ribosomes.

Inventors:
DESHAYES, Stephanie (6210 Canterbury Drive, #113Culver City, California, 90230, US)
WONG, Gerard C. L. (1521 Greenfield Avenue, Apt. 403Los Angeles, California, 90025, US)
KASKO, Andrea (11245 Malat Way, Culver City, California, 90230, US)
XIAN, Wujing (1521 Greenfield Avenue, Apt. 403Los Angeles, California, 90025, US)
SCHMIDT, Nathan (95 Behr Avenue, Apt. 302San Francisco, California, 94158, US)
Application Number:
US2017/060418
Publication Date:
May 11, 2018
Filing Date:
November 07, 2017
Export Citation:
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Assignee:
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA (1111 Franklin Street, Twelfth FloorOakland, California, 94607-5200, US)
International Classes:
A61K38/02; A61K47/55; A61P31/04; C07K2/00; C07K19/00
Domestic Patent References:
WO2016025627A12016-02-18
Other References:
SCHMIDT, NW ET AL.: "Engineering Persister-Specific Antibiotics with Synergistic Antimicrobial Functions", ACS NANO, vol. 8, no. 9, 18 August 2014 (2014-08-18), pages 8786 - 8793, XP055405416, DOI: doi:10.1021/nn502201a
DESHAYES, S ET AL.: "Designing Hybrid Antibiotic Peptide Conjugates To Cross Bacterial Membranes", BIOCONJUGATE CHEMISTRY, vol. 28, no. 3, 15 March 2017 (2017-03-15), pages 793 - 804, XP055605743, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.6b00725
Attorney, Agent or Firm:
WOOD, William J. (Gates & Cooper LLP, 6701 Center Drive WestSuite 105, Los Angeles California, 90045, US)
Download PDF:
Claims:
CLAIMS: 1. A composition of matter comprising:

a microbial membrane disrupting peptide comprising the sequence: GWIRNQFRKIWQR (SEQ ID NO: 1), GWRRNQFWIKIQR (SEQ ID NO: 2), GPWWFKWPRLI (SEQ ID NO: 3), GFHGVKNLARRIL (SEQ ID NO: 4), GWRNQIRKGWQR (SEQ ID NO: 5), CFHRLFKRILRK (SEQ ID NO: 6), RWWRLI (SEQ ID NO: 7), or RWRWIR (SEQ ID NO: 8); and

an antibiotic agent coupled to the microbial membrane disrupting peptide. 2. The composition of claim 1, wherein the antibiotic agent is an aminoglycoside, β-lactam, macrolide, quinolone, tetracycline, phenicol or sulfonamide. 3. The composition of claim 2, wherein the antibiotic agent is an aminoglycoside selected from a group consisting of tobramycin, neomycin, ampicillin,

aminopenicillin, amoxicillin, and kanamycin. 4. The composition claim 2, wherein the antibiotic agent is a β-lactam selected from a group consisting of penam, cephem, monobactam, and carbapenem. 5. The composition of claim 1, wherein the microbial membrane disrupting peptide consists of the sequence: GWIRNQFRKIWQR (SEQ ID NO: 1),

GWRRNQFWIKIQR (SEQ ID NO: 2), GPWWFKWPRLI (SEQ ID NO: 3),

GFHGVKNLARRIL (SEQ ID NO: 4), GWRNQIRKGWQR (SEQ ID NO: 5), CFHRLFKRILRK (SEQ ID NO: 6), RWWRLI (SEQ ID NO: 7) or RWRWIR (SEQ ID NO: 8).

6. The composition of claim 5, wherein the microbial membrane disrupting peptide is coupled to the antibiotic agent by an amino acid linker comprising 1-10 amino acids. 7. The composition of claim 1, wherein the antibiotic is coupled to the N- terminus of the peptide. 8. The composition of claim 1, wherein the antibiotic is coupled to the C- terminus of the peptide. 9. The composition of claim 1, further comprising a pharmaceutically acceptable carrier selected from at least one of: a pH adjusting agent, a buffering agents, a tonicity adjusting agent, a wetting agent, or a detergent. 10. An aminoglycoside-peptide conjugate composition comprising:

an aminoglycoside selected from a group consisting of tobramycin, neomycin, ampicillin, aminopenicillin, amoxicillin, and kanamycin; and

a microbial membrane disrupting peptide consisting essentially of an amino acid sequence selected from a group consisting of GWIRNQFRKIWQR (SEQ ID NO: 1), GWRRNQFWIKIQR (SEQ ID NO: 2), GPWWFKWPRLI (SEQ ID NO: 3), GFHGVKNLARRIL (SEQ ID NO: 4), GWRNQIRKGWQR (SEQ ID NO: 5), CFHRLFKRILRK (SEQ ID NO: 6), RWWRLI (SEQ ID NO: 7) or RWRWIR (SEQ ID NO: 8);

wherein:

the aminoglycoside is covalently conjugated to the microbial membrane disrupting peptide; and the aminoglycoside-peptide conjugate disrupts microbial membranes and inhibits microbial metabolism.

11. A method of inhibiting growth of a pathogenic bacteria comprising combining the pathogenic bacteria with a composition of any one of claims 1-10, wherein the composition is selected for its ability to inhibit the growth of the pathogenic bacteria. 12. The method of claim 11, wherein the pathogenic bacteria comprise anaerobic bacteria. 13. The method of claim 11, wherein the pathogenic bacteria are persisters exhibiting antibiotic agent resistance. 14. The method of claim 11, wherein the pathogenic bacteria is resistant to antibiotic agent or aminoglycoside not coupled to the microbial membrane disrupting peptide. 15. A method of synthesizing a multi-functional aminoglycoside-peptide conjugate comprising:

synthesizing a fully-protected peptide sequence with protected side chains using solid phase synthesis on a 2-chlorotrityl chloride resin;

protecting amine groups of an aminoglycoside with t-butyloxycarbony groups to provide a Boc-protected aminoglycoside;

selectively reacting a primary hydroxyl of the Boc-protected aminoglycoside with succinic anhydride to introduce a terminal carboxyl function;

coupling the Boc-protected aminoglycoside with the fully protected and resin- anchored peptide;

cleaving the conjugate off the resin; and

deprotecting the conjugate by treating the conjugate with a trifluoroacetic acid mixture containing scavengers; and

purifying the conjugate.

16. The method of claim 15, wherein the peptide comprises a peptide sequence selected from a group consisting of GWIRNQFRKIWQR (SEQ ID NO: 1),

GWRRNQFWIKIQR (SEQ ID NO: 2), GPWWFKWPRLI (SEQ ID NO: 3), GFHGVKNLARRIL (SEQ ID NO: 4), GWRNQIRKGWQR (SEQ ID NO: 5), CFHRLFKRILRK (SEQ ID NO: 6), RWWRLI (SEQ ID NO: 7) or RWRWIR (SEQ ID NO: 8). 17. The method of claim 16, wherein the antibiotic is an aminoglycoside selected from a group consisting of tobramycin, neomycin, ampicillin, aminopenicillin, amoxicillin, and kanamycin. 18. The method of claim 17, wherein the Boc-protected aminoglycoside is coupled with the N-terminal of the fully protected and resin-anchored peptide. 19. The method of claim 17, wherein the Boc-protected aminoglycoside is coupled with the C-terminal of the fully protected and resin-anchored peptide. 20. The method of claim 15, wherein the conjugate is purified using reversed- phase high performance liquid chromatography (HPLC).

Description:
MULTIFUNCTIONAL ANTIBIOTIC-PEPTIDE CONJUGATES:

SYNTHESIS AND ANTIMICROBIAL ACTIVITIES CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under Section 119(e) from U.S. Provisional Application Serial No. 62/418,549, filed November 7, 2016, entitled “MULTIFUNCTIONAL ANTIBIOTIC-PEPTIDE CONJUGATES: SYNTHESIS AND ANTIMICROBIAL ACTIVITIES”, the contents of which are incorporated herein by reference.

This application is related to co-pending U.S. Application No. 15/503,673 by Schmidt et al., that was filed in the U.S. Patent Office on February 13, 2017 entitled “MULTIFUNCTIONAL MEMBRANE-ACTIVE AMINOGLYCOSIDE-PEPTIDE CONJUGATES”, which is a national phase application of PCT Application No. PCT/US2015/044897, the contents of which are incorporated herein by reference. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant Nos. DMR1106106 and CHE-1112490 awarded by the National Science Foundation, and Grant No. 1-DP2-OD008533 awarded by the National Institutes of Health. The Government has certain rights in the invention. TECHNICAL FIELD

The invention relates to antimicrobials and antibiotics, in particular multifunctional antibiotics and methods for treating infections. BACKGROUND OF THE INVENTION

One significant health problem comes from the fact that bacterial communities almost always contain subpopulations of cells, known as persisters that are not susceptible to conventional antibiotics (Balaban NQ, et al. Science 2004;305(5690):1622-1625; Lewis K Nat Rev Microbiol 2007;5(1):48-56; Gefen O, et al. FEMS Microbiol Rev 2009;33(4):704-717). These bacteria are usually slow- growing or non-growing whose reduced metabolism allows them to survive antibiotic treatment, since most drugs target active growth processes (Lewis K Annual review of microbiology 2010;64:357-372). Once antibiotic treatment has ceased persisters can become active and renew infection, and continued exposure of bacteria to antibiotics increases the likelihood for the emergence of genetic resistance (Keren I, et al. FEMS Microbiol Lett 2004;230(1):13-18). Drug-tolerance is believed to contribute to chronic infections (Mulcahy LR, et al. Journal of bacteriology 2010;192(23):6191- 6199; Lewis K Annual review of microbiology 2010;64:357-372), and there are scarce potential therapeutic strategies against persistent bacteria (Allison KR, et al. Nature 2011;473(7346):216-220; Conlon BP, et al. Nature 2013;503(7476):365-370).

Thus, there is a need for compositions and methods of treating infections, in particular the treatment of persistent bacteria and other problems associated with antibiotic resistance. SUMMARY OF THE INVENTION

The present invention provides multifunctional antibiotic agents that exhibit enhanced antimicrobial activities against a variety of pathogens including difficult-to- kill persistent bacteria. The invention further provides methods for making and using these multifunctional antibiotic agents. As discussed below, the invention has a number of significant advantages over existing technologies in the art. These include: multiple modes of action in a single molecule; synergistic modes of action; the ability to eradicate pathogens that cannot be killed by conventional antibiotics; the ability to delay the emergence of bacterial resistance by killing persisters; deterministic and rational design rules; and flexibility to be tuned with different antibiotics and different peptide sequences a wide variety of therapeutic applications. Embodiments of the invention include those where traditional antibiotics are renovated or rejuvenated by combining them with peptides to produce a synergistic antimicrobial action in a single molecule. Typically, the invention provides a platform that combines the killing activity of traditional antibiotics (e.g. aminoglycosides, β-lactams) with the membrane permeation activity of an antimicrobial peptide so the resulting conjugate acts as an antimicrobial with increased killing activity compared to the parent antibiotic. This platform is tunable with different antibiotics and various peptide sequences based on the pre-established design rules described in PCT Application No. PCT/US2015/044897.

This work expands upon a previously described prototype agent, Pentobra, which is composed of a membrane-permeating peptide in combination with tobramycin, that demonstrated strong bactericidal activity against various persistent cells. The present invention describes additional compounds useful to treat chronic infections currently untreatable by conventional antibiotics and the synthesis of these new conjugates through various synthetic routes. The Example section below examines their antimicrobial activity through MICs assays (especially against multi- drug resistant strains such as Methicillin-resistant Staphylococcus aureus and Vancomycin-resistant Enterococci), killing assays, and biofilm inhibition assays.

The invention disclosed herein has a number of embodiments. Embodiments of the invention include a composition of matter comprising a microbial membrane disrupting peptide comprising the sequence: GWIRNQFRKIWQR (SEQ ID NO: 1), GWRRNQFWIKIQR (SEQ ID NO: 2), GPWWFKWPRLI (SEQ ID NO: 3), GFHGVKNLARRIL (SEQ ID NO: 4), GWRNQIRKGWQR (SEQ ID NO: 5), CFHRLFKRILRK (SEQ ID NO: 6), RWWRLI (SEQ ID NO: 7), or RWRWIR (SEQ ID NO: 8); and an antibiotic agent coupled to the microbial membrane disrupting peptide. In some embodiments of the invention, the antibiotic agent is an aminoglycoside, β-lactam, macrolide, quinolone, tetracycline, phenicol or sulfonamide. For example, in certain embodiments of the invention, the antibiotic agent is an aminoglycoside selected from a group consisting of tobramycin, neomycin, ampicillin, aminopenicillin, amoxicillin, and kanamycin. In other embodiments of the invention, the antibiotic agent is a β-lactam selected from a group consisting of penam, cephem, monobactam, and carbapenem. Optionally in these embodiments, the microbial membrane disrupting peptide is coupled to the antibiotic agent by an amino acid linker (e.g. one comprising 1-10 amino acids). Typically, the antibiotic is coupled to the N-terminus or the C-terminus of the peptide. Such compositions can further comprise a pharmaceutically acceptable carrier selected from at least one of: a pH adjusting agent, a buffering agents, a tonicity adjusting agent, a wetting agent, or a detergent.

As noted above, illustrative embodiments if the invention include aminoglycoside-peptide conjugate compositions. These compositions can include an antibiotic such as an aminoglycoside selected from a group consisting of tobramycin, neomycin, ampicillin, aminopenicillin, amoxicillin, and kanamycin, and a microbial membrane disrupting peptide coupled to this antibiotic. The microbial membrane disrupting peptide typically consist of an amino acid sequence disclosed herein such as one selected from a group consisting of GWIRNQFRKIWQR (SEQ ID NO: 1), GWRRNQFWIKIQR (SEQ ID NO: 2), GPWWFKWPRLI (SEQ ID NO: 3), GFHGVKNLARRIL (SEQ ID NO: 4), GWRNQIRKGWQR (SEQ ID NO: 5), CFHRLFKRILRK (SEQ ID NO: 6), RWWRLI (SEQ ID NO: 7) or RWRWIR (SEQ ID NO: 8). In one instance, the antibiotic is an aminoglycoside and the conjugate functions as a metabolic inhibitor by binding to bacterial ribosomes via the aminoglycoside. In a further instance, the membrane disruptive capability causes the membrane of a bacteria to become permeable to the conjugate. The antibiotic may be conjugated to the peptide using amide bond formation, succinimide coupling, reductive amination,“click” chemistry or thiol-maleimide coupling. The antibiotic may be conjugated to the N-terminal or C-terminal of the peptide. In one specific instance, the antibiotic is conjugated to the C-terminal of the peptide through a 1,2,3- triazole ring using“click” chemistry. Other embodiments of the invention provide a method of synthesizing a multi- functional aminoglycoside-peptide conjugate. The method comprises synthesizing a fully-protected peptide sequence with protected side chains using solid phase synthesis on a 2-chlorotrityl chloride resin. Amine groups of an aminoglycoside are protected with t-butyloxycarbony groups to provide a Boc-protected aminoglycoside. A primary hydroxyl of the Boc-protected aminoglycoside is selectively reacted with succinic anhydride to introduce a terminal carboxyl function. The Boc-protected aminoglycoside is coupled with the fully protected and resin-anchored peptide. The Boc-protected aminoglycoside may be coupled with the N-terminal or C-terminal of the fully protected and resin-anchored peptide. The conjugate is then cleaved off the resin and deprotected by treating the conjugate with a trifluoroacetic acid mixture containing scavengers. The conjugate may be further purified using a technique such as reversed-phase high performance liquid chromatography (HPLC).

In other embodiments of the invention, a method of treating a bacterial infection (e.g. an infection in a mammal by a pathogenic bacteria) is provided. In certain embodiments, the bacteria of the bacterial infection are persisters, bacteria which exhibit resistance to conventional therapeutic regimes with antibiotic agents. The method comprises administering to the mammal a therapeutically effective amount of an antibiotic-peptide conjugate described herein. In certain instances, the bacterial infection is resistant to the unconjugated antibiotic. In some instances the bacterial infection comprises anaerobic bacteria.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-C provide examples of conjugates composed of a membrane-active peptide attached to ampicillin through a maleimide group, in accordance with one or more embodiments of the invention.

FIGS. 2A-C provides graphs depicting single micromolar concentrations of Pentobra and cPentobra reduced stationary (FIG. 2A) and persistent (FIG. 2B) P. aeruginosa PA14 cells. Tobramycin is much less effective. No synergistic effect is observed when the Pen peptide is mixed with tobramycin. (FIG. 2C) Pentobra and cPentobra inhibit P. aureginosa biofilm growth while tobramycin has negligible effect.

FIGS.3A-B provide a schematic illustrating the synthesis of cPentobra. (FIG. 3A) Synthesis of Fmoc-Pra(BocTobra)-OH. (FIG. 3B) Grafting of Fmoc- Pra(BocTobra)-OH to 2-chlorotrityl chloride resin, followed by peptide synthesis on solid support using Fmoc strategy, and final cleavage and deprotection of the peptide using a mixture of trifluoroacetic acid (TFA) and scavengers. The gray sphere represents the 2-chlorotrityl chloride resin.

FIGS. 4A-B provide graphs depicting membrane permeabilization of MAAPCs. A) E. coli D31 outer membrane permeabilization by MAAPCs; B) E. coli ML35 inner membrane permeabilization by MAAPCs. The concentration for all tested compounds is 2 μM. DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. In the description of the preferred embodiment, reference may be made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

The present invention provides multifunctional antibiotics that address the problem of chronic and recurrent infections that are typically caused by bacterial “persisters”. Persisters are non-growing, dormant cells capable of surviving antimicrobial treatment. Cell cultures almost always contain subpopulations of persister cells. Once the treatment is ceased, population growth resumes causing a relapsing chronic infection such as Staphylococcus aureus infections implicated in prosthetic joints or Pseudomonas aeruginosa pulmonary infections in patients with cystic fibrosis. Unlike resistance, persistence is a non-heritable phenotype. However, increasing evidence suggests that persisters may serve as an evolutionary reservoir from which resistance might emerge.

Rather than providing a drug that attempts to kill pathogens that are already resistant, the present invention prevents the emergence of resistance by eradicating these persistent cells before they represent a pool of adaptively evolving organisms from which resistant mutants can emerge. This angle of attack offers a two-in-one approach by both inhibiting the spread of resistance and treating chronic infections by killing persistent cells. This approach is based on the observation that persister cells have reduced metabolism leading to a defect in drug uptake. Therefore, disrupting the cell membrane in a manner similar to the activity of antimicrobial peptides (AMP) provides an alternative pathway for a drug to penetrate into these cells and reach the cellular machinery. One important feature of the present invention is the capability of the multifunctional antibiotic to defeat bacteria that are refractory to traditional antibiotic therapies through multiple and synergistic modes of action. This provides a significant competitive advantage in a sector of the healthcare industry, which is in dire need of new therapeutics for the treatment of recurrent and chronic infections. While large pharmaceutical companies are the major players engaged in the development of antibiotics, most of them have scaled back or dropped their anti- infectives discovery. Thus, direct market competition is mostly in the form of small start-ups. However, these biotechs are exploring either a“cocktail” approach (in which multiple molecules are combined but not chemically conjugated) or have developed new molecules with a single mode of action that act on a specific target in bacteria, thus ultimately the chances for resistance to emerge are still high. The present invention provides an approach that is totally different from the existing ones because it combines multiple antibiotic functions in a single molecule, and therefore has strong advantages over cocktails or single function antibiotics. Unlike most approaches that are serendipitous, the present approach is deterministic and rational as the drug design is based on specific rules that selectively induce permeabilization of bacterial membranes (see PCT Application No. PCT/US2015/044897).

In an illustrative embodiment, membrane-active antibiotic-peptide conjugates (MAAPCs) are multi-functional antibiotics that (1) exhibit bactericidal properties by functioning as metabolic inhibitors, including by binding to bacterial ribosomes via the aminoglycoside component of the conjugate; and (2) exhibit membrane disruptive capability (e.g., membrane permeabilization ability such as antimicrobial peptide (AMP) or cell penetrating peptide (CPP) activities) by virtue of the peptide component of the conjugate, which acts to render the whole aminoglycoside-peptide conjugate membrane-disruptive (see PCT Application No. PCT/US2015/044897, filed 08/2015). Table 1 and Figure 1 show various conjugates synthesized through different synthetic routes using different classes of antibiotics. One important feature of the conjugate is its capability to defeat bacteria that are refractory to traditional antibiotic therapies through multiple and synergistic modes of action. This approach increases the activity spectrum of not only the class of aminoglycosides but also other classes of antibiotics, and may also delay the emergence of bacterial resistance.

In other illustrative embodiments, MAAPCs 01-10 as shown in the Tables below are synthesized based on a similar method used for the prototype Pentobra [1, 2], but with different peptide sequences and/or different aminoglycosides (e.g. tobramycin, neomycin and kanamycin). In addition, an analogue of Pentobra named cPentobra (MAAPC07) is synthesized using a different synthetic approach. While the same peptide sequence and same aminoglycoside (tobramycin) are used as for Pentobra, tobramycin is conjugated to the C-terminal of the peptide through a 1,2,3- triazole ring using“click” chemistry (Figure 3).

Other aminoglycosides that may be used include, but are not limited to, amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin, fradiomycin, gentamicin, ispamicin, kanamycin, micronomicin, neomycin A, neomycin B, neomycin C, neomycin E, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, and tobramycin, including pharmaceutically acceptable salts and esters thereof. The approach provided herein is also not limited to aminoglycosides and may be used to renovate other classes of antibiotics such as β- lactams (e.g. penams, cephems, monobactams, carbapenems), macrolides, quinolones, tetracyclines, phenicols and sulfonamides. Figure 1 shows some examples of conjugates composed of a membrane active peptide (pre-defined according to our design rules, see PCT Application No. PCT/US2015/044897, filed 08/2015) attached to ampicillin (or other aminopenicillins such as amoxicillin) using a thiol-maleimide coupling. For those examples, an extra cysteine (bearing a thiol on its side chain) is incorporated either at the N-terminal or C-terminal or between two amino acids of the peptide sequence; concurrently, ampicillin is modified with a maleimide group on its primary amine to allow a thiol-maleimide coupling with the cysteine-modified peptide. This list is not exhaustive and can be extended to other antibiotics using different spacers, linkers and synthetic approaches. The coupling reactions include known methods in the art, such as amide bond formation (e.g., via succinimide coupling), reductive amination,“click” chemistry, thiol-maleimide coupling at either the N-terminal or C-terminal ends of the peptide after modification with appropriate functionality of both, the peptide and the antibiotic. Coupling can take place either in solution or solid phase.

Embodiments of the presently disclosed invention relate to antibiotic-peptide conjugates that comprise an and antibiotic such as an aminoglycoside (A) covalently coupled, typically via a linker moiety (L), to a peptide (P), according to a general formula P-L-A as described herein. While aminoglycosides are used as an example in the following text, other antibiotics can be adapted for use with embodiments of the invention. In such embodiments, the aminoglycoside and the peptide are selected according to a design principle such that the conjugate as a whole possesses specific charge and hydrophobicity properties that permit it to permeate the plasma membranes of target bacterial cells and to impair vital ribosomal function in such cells.

The herein disclosed aminoglycoside-peptide conjugates unexpectedly exhibited superior bactericidal properties relative to unconjugated aminoglycosides or free peptides (e.g., membrane-disrupting peptides such as antimicrobial peptides (AMP) or cell penetrating peptides (CPP)) and in particular, surprisingly provided such bactericidal properties without interference by the peptide moiety with the activity of the aminoglycoside moiety, and without interference by the aminoglycoside moiety with the activity of the peptide moiety. As also described herein, the present conjugates were effective bactericides against exemplary antibiotic-tolerant and antibiotic-resistant bacterial strains even where the conjugate comprised the same aminoglycoside moiety to which the antibiotic-resistant bacteria had become resistant, a result that could not have been predicted. The peptide-linker- aminoglycoside conjugate is thus a dual action antimicrobial compound that is both membrane-active and ribosomally active. As discussed in further detail below, the conjugates disclosed herein overcome the limitations of the aminoglycoside antibiotics in the current state of the art. Presently disclosed embodiments will find uses in the treatment of bacterial infections, including in the treatment of antibiotic- resistant bacteria. The presently disclosed conjugate operates as a membrane-active aminoglycoside drug that, unlike previously described aminoglycosides, has membrane activity by which it can facilitate its own uptake into cells. This means the drug is not reliant on energy-dependent internalization mechanisms such as proton- motive force (PMF) generation. The presently described membrane-active aminoglycoside conjugates will therefore have bactericidal activity against bacterial cells regardless of the metabolic state of the bacterial cells. Accordingly, it is believed by way of non-limiting theory that the present conjugates have an activity spectrum that extends to all targeted bacterial cells, including sub-populations of persisters. Anaerobic bacteria should also be vulnerable to the present membrane- active aminoglycoside conjugates, since the conjugates self-promote cellular uptake by membrane permeabilization. By judicious choice of peptide sequence (as described below), the present composite aminoglycoside-peptide conjugate molecule can be designed to act as a molecular transporter, e.g., as a vector that can cross bacterial membranes and deliver bioactive cargo into cells. In the case of intracellular pathogens like M. tuberculosis, the present membrane-active aminoglycoside conjugate may according to certain embodiments be engineered to penetrate host macrophage membranes, thereby allowing the aminoglycoside to reach intracellular bacilli.

Advantageously, the present membrane-active aminoglycoside (P-L-A conjugate) has multiple mechanisms of action, which may overcome typical mechanisms of antibiotic resistance in bacteria and thereby render less likely the advent of bacterial resistance to the aminoglycoside moiety than would be the case when free aminoglycoside is administered. The robust membrane activity built into the present membrane-active aminoglycoside conjugate will thus limit the viability of bacterial resistance strategies such as those that relate to increasing cell wall and membrane impermeability. Resistance mechanisms that render aminoglycosides inert or ineffective from drug or target site alterations will not abolish activity, since the present membrane-active aminoglycoside conjugate (P-L-A) will retain a baseline level of antimicrobial activity from an auxiliary mechanism of action via its ability to promote membrane disruption. Additionally and in certain embodiments, where the present membrane-active aminoglycoside antibiotic (P-L-A conjugate) comprises a recombinant peptide linked to an aminoglycoside to provide a hybrid molecule, the conjugate represents a modified substrate for which the bacterial resistance mechanisms, such as aminoglycoside disabling enzymes and efflux pumps, may have reduced affinities. The present conjugates will thus find uses for overcoming antibiotic resistant bacteria.

The present membrane-active aminoglycoside P-L-A conjugate may also advantageously provide multiple mechanisms of selectivity for additional safety. For example, in addition to the natural selectivity exhibited by aminoglycosides for bacterial ribosomes, the present membrane-active aminoglycosides (P-L-A) conjugates preferentially permeate bacterial membranes, a property that is shared by innate immunity peptides, but which is not a capability of aminoglycoside antibiotics per se.

In addition, the presently disclosed membrane-active aminoglycoside conjugates will beneficially reduce the duration, dose and/or frequency of aminoglycoside administration to a subject (e.g., a patient), which are among the recognized significant risk factors for aminoglycoside toxicity. In this regard, the present membrane-active aminoglycoside conjugates may be optimized according to the drug design principles described herein, providing superior efficacies than free aminoglycosides, and decreasing the chance of recurrent infection since they have multiple mechanisms of action. Increased drug potency of the present conjugates is believed, by way of non-limiting theory, to provide an equivalent or superior therapeutic index than is possible with previous aminoglycoside regiments, through lower drug dosages and shorter treatment durations. As multi-functional drugs in view of the membrane-disrupting and ribosome-inhibiting functionalities provided by the present P-L-A conjugates, bactericidal activity against a greater proportion of the target bacterial cell population is provided, thereby reducing the incidence of recurrent infections from persistent bacteria.

In certain embodiments disclosed herein for the first time, compositions are provided that comprise the presently described multifunctional membrane-active aminoglycoside peptide (P-L-A) conjugate and that further comprise an adjuvant moiety as described in greater detail below. The adjuvant moiety, which may comprise at least one saccharide compound as provided herein and/or at least one glycomimetic adjuvant moiety as provided herein, surprisingly confers a synergizing (i.e., the bactericidal activity of the conjugate when combined with the adjuvant is greater, in a statistically significant manner, than the sum of the bactericidal activities of the conjugate and the adjuvant when each component is separately contacted with target bacteria) capability on the composition that was unexpected and could not have been predicted.

As noted above and according to non-limiting theory, the present conjugates may be capable of self-promoting metabolic energy-independent cellular uptake by membrane permeabilization and are thus believed to provide unprecedented efficacy against antibiotic-resistant bacteria such as anaerobic and/or persister cells. Without wishing to be bound by theory, the role of PMF in aminoglycoside uptake into bacterial cells is thought to be responsible for weak aminoglycoside activity against dormant bacteria, or persisters, a sub-population which has been implicated as a major source of antibiotic treatment failure and drug resistance. Empirically, anaerobic bacteria are less susceptible to aminoglycoside antibiotics. Since many of the energy- dependent processes that establish a PMF use oxygen, the intrinsic resistance of anaerobic bacteria to aminoglycosides has been attributed to reduced aminoglycoside uptake. Also, the inability of aminoglycosides to cross membranes independently reduces their effectiveness against intracellular pathogens like Mycobacterium tuberculosis, which invade and replicate in host macrophages.

Additionally, however, certain embodiments are presently contemplated in which the P-L-A conjugate is present along with an adjuvant moiety that is capable of advantageously synergizing with or enhancing (e.g., increasing in a statistically significant manner relative to the sum of the levels of activity that are present using the P-L-A conjugate and the adjuvant moiety separately) bactericidal activity of the conjugate in what is believed, further according to non-limiting theory, to be a process that is specific to the herein disclosed compositions comprising membrane-active aminoglycoside peptide conjugates and adjuvant moieties. The presently described compositions comprising a synergizing P-L-A conjugate and an adjuvant moiety (e.g., a saccharide compound or a glycomimetic adjuvant moiety as provided herein) surprisingly exploit adjuvant-stimulated conjugate uptake by bacterial cells such as persister cells even where the present conjugates are shown not to depend strongly on the metabolic status of the cell, and thus would not have been expected to synergize when combined with the adjuvant. Accordingly, in certain embodiments, the aminoglycoside-peptide conjugate is represented by the following general formula:

P-L-A in which:

A comprises an antibiotic such as an aminoglycoside;

P comprises a peptide; and

L comprises a linker moiety that covalently couples the aminoglycoside to the peptide; wherein:

(a) the conjugate has a total charge Q T of from 3 + to 10 + based on number of amine groups in A, number of lysine, arginine and histidine amino acids in P, and number of aspartic acid and glutamic acid amino acids in P, wherein

Q T = (N A + N P )– (N DE ) in which

N A is the number of amine groups in A,

N P is the number of lysine, arginine and histidine amino acids in P,

N DE is the number of aspartic acid and glutamic acid amino acids in P,

(b) the conjugate has a composite octanol:water partition coefficient (Log P) conjugate of from -1.5 to zero, and

(c) P comprises a peptide of at least 6 amino acids and not more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids in which

(i) N DE is less than or equal to 2,

(ii) at least 25% of the amino acids are hydrophobic amino acids that are each independently selected from phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine, wherein optionally, when P consists of n amino acids of which at least z amino acids are hydrophobic and 25% of n has a non- integer value, then z is an integer having a maximum whole number value that is less than the non-integer value that is 25% of n (for example, if n = 6, 25% of n is 1.5 such that z = 1, the maximum whole number value that is less than 1.5; hence at least one amino acid of P is hydrophobic),

(iii) the peptide has an average Eisenberg Consensus scale hydrophobicity Φ of from -0.6 to 0.2 wherein in which P consists of n amino acids and w i is Eisenberg Consensus scale hydrophobicity of the i th amino acid.

The various components of the aminoglycosides-peptide conjugates are described in detail below. Aminoglycosides

The aminoglycoside (A) portion of the herein disclosed peptide-linker- aminoglycoside (P-L-A) conjugate is ribosomally active once the conjugate permeates through the bacterial cell membrane. In addition, the aminoglycoside contributes to the overall charge and hydrophobicity of the conjugate, thereby influencing the cell permeation behavior as well.

As used herein, an aminoglycoside refers to an organic molecule derived at least in part from a saccharide or polysaccharide. Typically, an aminoglycoside contains two or more aminosugars linked by glycosidic bonds to an aminocyclitol component. For instance, the aminoglycosides are oligosaccharides consisting of an aminocyclohexanol moiety glycosidically linked to other amino sugars. An aminoglycoside may be either a synthetic or natural antibiotic, such as aminoglycoside antibiotics that may be isolated from species of Streptomyces and Micromonospora, but in preferred embodiments of the herein disclosed conjugate of general formula P-L-A, the aminoglycoside has been modified to obtain a non- naturally occurring molecule once the peptide and linker moieties have been incorporated into the conjugate.

Aminoglycosides include, without limitation, amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), fradiomycin, gentamicin, ispamicin, kanamycin, micronomicin, neomycins (A, B, C and E forms), neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, streptonicozid, and tobramycin, including pharmaceutically acceptable salts and esters thereof.

Any aminoglycoside employed herein, in isolation or coupled to a peptide, comprises one or more free amino groups, including a primary amino group represented by–NH 2 , and a secondary amino group represented by–NHR (wherein R is an alkyl group. The amino group may be protonated and thus bears a positive charge. The number of the free amino groups in a given aminoglycoside determines the charges of the aminoglycoside portion of the conjugate. The aminoglycoside, accordingly to the various embodiments disclosed herein, may be coupled to a peptide by any reactive functional group that may be present on the aminoglycoside, including without limitation, hydroxyl (primary and secondary), amino group (primary and secondary amino group), aldehyde, and the like.

In preferred embodiments, the aminoglycoside further comprises a primary hydroxyl moiety, which is typically on carbon 6 of an aminosugar or glucose. The primary hydroxyl moiety has a higher relative reactivity (compared to secondary hydroxyls). Additionally, the primary hydroxyl group of various aminoglycosides including tobramycin is reportedly not essential for RNA binding. Thus, the primary hydroxyl group is a convenient point of attachment to a linker moiety without the risk of compromising the ribosomal activity of the aminoglycoside. Peptides

The peptide (P) portion, which may also be referred to as a“membrane- disruptive peptide,” is designed based on global trendlines previously developed for known antimicrobial peptides (AMPs) or cell penetration peptides (CPP).

Antimicrobial peptides (AMPs) were discovered in the 1980s when a family of short peptides with broad spectrum antimicrobial activity was isolated from frogs. Since then well over 1000 AMPs have been discovered in a broad range of plant and animal species. Collectively, AMPs have broad spectrum antimicrobial activity and have a rapid mechanism of action. Unlike traditional antibiotics that are in clinical use like aminoglycosides, which have core structural features that are primarily responsible for their antimicrobial activities, AMP sequences are highly diverse and do not share a core structure. Instead, AMPs share a common motif: they are cationic and amphipathic. From in vitro studies, the general mechanism of AMP activity is believed to involve the selective disruption and permeabilization of bacterial membranes.

Synthetic AMPs have been developed by modifying natural versions, and by creating new AMP analogues. Examples of synthetically derived AMPs include pexiganan, a variant of the AMP magainin from frogs; iseganan, a variant of protegrin from pigs; and omiganan, a cattle indolicidin variant. (For review see Hancock et al., Nat. Biotech. (2006) 24, 1551-1557). Synthetic AMP analogues have been constructed by mimicking the peptide primary structure (peptidomimetics), and using non-peptidic compounds. Peptidomimetic AMPs include designing amphiphilic mimics through modification of the peptide backbone using β-peptides and peptoids, and D-amino acid substitutions. Examples of non-peptidic compounds are amphiphilic oligomers with phenylene ethynylene, short polymethacrylate, and polynorbornene backbones (see, e.g., Scott et al., Curr. Opin. Biotech. (2008) 19, 620- 627 and references therein). While many of these AMP constructs have displayed potent in vitro activity against a number of bacterial pathogens, to date, AMP-based antibiotics are not in clinical use. Daptomycin, which is from the soil bacteria streptomyces, is currently the only FDA-approved antibiotic with a primary mechanism of action from membrane activity.

AMPs have broad spectrum antimicrobial activity and have a rapid mechanism of action. AMP sequences are highly diverse and do not share a single core structure. Instead, AMPs share a common motif: they are cationic and amphipathic. From in vitro studies, AMP sequences, CPP sequences, and non-peptidic membrane-active sequences were found to generate the nanoscopic membrane curvature necessary for permeation. Thus, the general mechanism of AMP activity is believed to involve the selective disruption and permeabilization of bacterial membranes. Global trendlines were developed by examining how patterns in the cationic and hydrophobic compositions of the AMPs relate to the geometric requirements of membrane topology changes. Mishra et al, Proc. Natl. Acad. Sci. U. S. A. 108, 16883-16888 (2011); Schmidt et al, J. Am. Chem. Soc.133, 6720-6727 (2011); Schmidt et al, J. Am. Chem. Soc. 134, 19207-19216 (2012); Hu et al, Macromolecules 46, 1908-1915 (2013).

Although AMP antimicrobial activity is typically lower than that of aminoglycosides, AMP membrane activity depends less on the metabolic status of the cell. Hurdle et al, Nat. Rev. Microbiol. 9, 62-75 (2011). Previous work has shown that tobramycin can retain good activity after conjugation to lipid tails (see Bera, S. et al, J. Med. Chem.51, 6160-6164 (2008); Dhondikubeer, R. et al. J. Antibiot.65, 495- 498 (2012)). However, prior to the present disclosure there has been no general methodology for combining two distinct antimicrobial functions into a single molecule without mutual interference.

The aminoglycoside-peptide conjugates described herein can result from a design rule that programs cell penetrating activity into an aminoglycoside by leveraging its free amine groups and adding a peptide sequence to generate a multifunctional antibiotic that combines membrane-penetrating activity with inhibition of protein synthesis. In particular, the aminoglycoside and the peptide are selected according to the following design rule to generate a conjugate that attains specific net charges and hydrophobicity to achieve membrane permeation.

First, an aminoglycoside is chosen and a site is determined on the molecule at which to link a short peptide (e.g., a peptide of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids). The site of conjugation should interfere minimally with the ribosomal binding function of the aminoglycoside. The aminoglycoside may, by way of non-limiting example, be any of the natural amino- modified sugar antibiotics from the aminoglycoside family and trivial variants including, but not limited to tobramycin, kanamycin, amikacin, gentamicin, neomycin, paromomycin, streptomycin, dibekacin, sisomicin, or netilmicin.

Second, determinations are made of (i) the number of protonated groups on the aminoglycoside (typically +3 to +5) at the relevant physiological pH, and (ii) the Log P value of the aminoglycoside, which is the octanol to water partition coefficient. Protonation states and Log P values for many aminoglycosides are published. Here P ≡ mole fraction partition coefficient of solute in octanol to that in water, so Log P = log{([solute] oct /[solute] wat )(V oct /V wat )} where V oct /V wat is the ratio of molar volumes of octanol and water. This can be converted to a free energy of transfer (ΔG) from octanol to water using the following formula: ΔG = -2.3 RT Log P, where RT = 0.5925 kcal/mol. The octanol→water partition coefficient Log P values for aminoglycosides are tobramycin, -3.41, gentamycin, -2.47, kanamycin, -4.55, neomycin, -3.65, streptomycin, -2.53, amikacin, -3.34, paromomycin, -3.27.

Third, the amine content and hydrophobicity (as measured by Log P) of the aminoglycoside are compared to the global trendline for AMP or CPP sequence compositions based on saddle-splay curvature selection rules. This is most easily accomplished by overlaying the position of the aminoglycoside on the saddle-splay curvature graphs for AMPs/CPPs (e.g., as described in Mishra et al, Proc. Natl. Acad. Sci. U. S. A. 108, 16883-16888 (2011); Schmidt et al, J. Am. Chem. Soc. 133, 6720- 6727 (2011); Schmidt et al, J. Am. Chem. Soc. 134, 19207-19216 (2012); Hu et al, Macromolecules 46, 1908-1915 (2013)), to compare its position with the global trendlines for AMPs and CPPs. The saddle-splay curvature graphs plot the average hydrophobicity of a peptide versus the number of lysines (N K ) plus the number of histidines (N H ) in the peptide divided by the total number of cationic amino acids in the peptide (N K +N H )/(N K +N H +N R ) where K, H, and R are lysine, histidine, and arginine, respectively (N R = number of arginines). Average hydrophobicity is calculated by taking the linear sum of the hydrophobicities of each amino acid in the sequence and dividing this value by sequence length. Hydrophobicity values for amino acids are determined from established scales such as Eisenberg consensus, Wimley-White, and Kyte-Doolittle hydrophobicity scales. Procedures for converting the Log P of an aminoglycoside to a hydrophobicity value (or vice versa) are published (e.g., Macromolecules (2013) 46:1908; Biochemistry (1996) 35:5109).

In preferred embodiments, the peptide (P) has an average Eisenberg Consensus scale hydrophobicity Φ of from -0.6 to 0.2 wherein

in which P consists of n amino acids and w i is Eisenberg Consensus scale hydrophobicity of the i th amino acid. The hydrophobicities of the 20 canonical amino acids according the Eisenberg consensus scale (Faraday Symp. Chem. Soc., (1982) 17:109-120) are: Ile, 0.73, Phe, 0.61, Val, 0.54, Leu, 0.53, Trp, 0.37, Met, 0.26, Ala, 0.25, Gly, 0.16, Cys, 0.04, Tyr, 0.02, Pro, -0.07, Thr, -0.18, Ser, -0.26, His, -0.40, Glu, -0.62, Asn, -0.64, Gln, -0.69, Asp, -0.72, Lys, -1.1, Arg, -1.8.

Determining the (N K +N H )/(N K +N H +N R ) for the peptide-linker-aminoglycoside (P-L-A) conjugate molecule is straightforward: N H and N R are, respectively, the number of positively charged histidine and arginine amino acids in the peptide, and N K is the number of lysines plus the number of protonated amines in the aminoglycoside.

Accordingly, in preferred embodiments the conjugate has a total charge Q T of from 3 + to 10 + based on number of amine groups in A, number of lysine, arginine and histidine amino acids in P, and number of aspartic acid and glutamic acid amino acids in P, wherein

in which N A is the number of amine groups in A, N P is the number of lysine, arginine and histidine amino acids in P, and N DE is the number of aspartic acid and glutamic acid amino acids in P; the peptide preferably comprises at least 6 amino acids and not more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids in which (i) N DE is less than or equal to 2, and (ii) at least 25% of the amino acids are hydrophobic amino acids that are each independently selected from phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine. With respect to certain embodiments in which at least 25% of the amino acids are hydrophobic amino acids that are each independently selected from phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine, it is contemplated that when P consists of n amino acids of which at least z amino acids are hydrophobic and 25% of n has a non-integer value, then z is an integer having the maximum whole number value that is less than the non-integer value that is 25% of n (for example, if n = 6, 25% of n is 1.5 such that z = 1, the maximum whole number value that is less than 1.5; hence at least one amino acid of P is hydrophobic).

In certain other preferred embodiments the conjugate has a total charge Q T of from 3 + to 10 + based on number of amine groups in A, number of lysine, arginine and histidine amino acids in P, and number of aspartic acid and glutamic acid amino acids in P, wherein

in which N A is the number of amine groups in A, N P is the number of lysine, arginine and histidine amino acids in P, and N DE is the number of aspartic acid and glutamic acid amino acids in P; the peptide preferably comprises at least 6 amino acids and not more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids in which (i) N DE is less than or equal to 2, and (ii) at least 25% of the amino acids are hydrophobic amino acids that are each independently selected from phenylalanine, isoleucine, leucine, methionine, valine, tryptophan, and tyrosine.

The hydrophobicity of the conjugate molecule is determined by molecular weight (MW) weighted average of the hydrophobicities of the peptide (P) and the aminoglycoside (A): Hydrophobicity = (MWP(Hydrophobicity of P) + MWA(Hydrophobicity of A))/(MWP + MWA). In preferred embodiments the conjugate has a composite octanol:water partition coefficient (Log P)conjugate of from -1.5 to zero. Log P is defined above. Using the Wimley-White water to octanol scale for free energies of transfer of the 20 canonical amino acids (Biochemistry (1996), 35:5109-5124) their Log P values are: Ala, -0.3669, Cys, 0.0157, Asp, -2.6711, Glu, -2.6637, Phe, 1.2548, Gly, -0.8439, His, -1.7098, Ile, 0.8219, Lys, -2.0547, Leu, 0.9173, Met, 0.4917, Asn, -0.6237, Pro, - 0.1027, Glu, -0.5650, Arg, -1.3282, Ser, -0.3376, Thr, -0.1835, Val, 0.3376, Trp, 1.5337, Tyr, 0.5210.

The composite octanol→water partition coefficient, Log P, for the peptide- aminoglycoside conjugate molecule is preferably from -1.5 to zero and is defined as the molecular weight (MW) weighted average of the Log P of the peptide (PEP) and aminoglycoside (AG):

where n = the number of amino acids in the peptide, and LogPi = the Log P of the i th amino acid in the peptide.

In certain embodiments the conjugate is covalently linked to an adjuvant moiety as provided and described herein. In these instances the physicochemical properties of the adjuvant will contribute to the overall nature of the composite conjugate-adjuvant molecule. In preferred embodiments the conjugate-adjuvant molecule has total charge QT of from 3 + to 10 + based on the number of amine groups in A, the number of lysine, arginine and histidine amino acids in P, the number of aspartic acid and glutamic acid amino acids in P, and the sum of the charged groups in the adjuvant, S, wherein

in which NA is the number of amine groups in A, NP is the number of lysine, arginine and histidine amino acids in P, and NDE is the number of aspartic acid and glutamic acid amino acids in P, and NS is the sum of the positive and negative charges in S. The hydrophobicity of the conjugate-adjuvant molecule is determined by molecular weight (MW) weighted average of the hydrophobicities of the peptide (P), aminoglycoside (A), and adjuvant (S): Hydrophobicity = (MWP(Hydrophobicity of P) + MWA(Hydrophobicity of A) + MWS(Hydrophobicity of S))/(MWP + MWA + MWS). The composite octanol→water partition coefficient, Log P, for the peptide- aminoglycoside-adjuvant molecule is preferably from -1.5 to zero and is defined as the molecular weight (MW) weighted average of the Log P of the peptide (PEP), aminoglycoside (AG), and adjuvant (ADJ):

Where the Log P of the peptide is defined above, and the Log P of the adjuvant is:

where n = the number of sugar monomers in the adjuvant, and LogPi = the Log P of the i th sugar in the adjuvant. The octanol→water partition coefficient Log P values for sugars are known in the literature (for example, Mazzobre et. al., Carbohydrate Research (2005) 340, 1207-1211) for glucose, -2.82, sucrose, -3.30, trehalose, -3.77.

The terms "polypeptide" "protein" and "peptide" and "glycoprotein" are used interchangeably and mean a polymer of amino acids not limited to any particular length. The term does not exclude modifications such as myristylation, sulfation, glycosylation, phosphorylation, formylation, and addition or deletion of signal sequences. The terms "polypeptide" or "protein" means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non- covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non- recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. Thus, a "polypeptide" or a "protein" can comprise one (termed "a monomer") or a plurality (termed "a multimer") of amino acid chains. The terms “peptide,” "polypeptide" and "protein" specifically encompass the peptides of the present disclosure, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acid of a herein described peptide.

The term“isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polypeptide or nucleic acid present in a living animal is not isolated, but the same polypeptide or nucleic acid, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term“gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region“leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).

The terms "isolated protein" and“isolated polypeptide” referred to herein means that a subject protein or polypeptide (1) is free of at least some other proteins or polypeptides with which it would typically be found in nature, (2) is essentially free of other proteins or polypeptides from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a protein or polypeptide with which the "isolated protein" or“isolated polypeptide” may be associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein or polypeptide can be encoded by genomic DNA, cDNA, mRNA or other RNA, may be of synthetic origin according to any of a number of well known chemistries for artificial peptide and protein synthesis, or any combination thereof. In certain embodiments, the isolated protein or polypeptide is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

The term "polypeptide fragment" refers to a polypeptide, which can be monomeric or multimeric, that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion or substitution of a naturally-occurring or recombinantly-produced polypeptide. As used herein,“contiguous amino acids” refers to covalently linked amino acids corresponding to an uninterrupted linear portion of a disclosed amino acid sequence. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 6 to about 100 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids long.

Certain preferred embodiments contemplate wholly artificial chemical synthesis of the herein described peptide moieties according to any of a number of established methodologies, such as those described in Amino Acid and Peptide Synthesis (Jones, J., 2002 Oxford Univ. Press USA, New York), Ramakers et al. (2014 Chem. Soc. Rev. 43:2743), Verzele et al. (2013 Chembiochem. 14:1032), Chandrudu et al. (2013 Molecules 18:4373), and/or Mäde et al. (2004 Beilstein J. Org. Chem. 10:1197). For example, manual or preferably automated solid-phase peptide synthesis based on the Merrifield method or other solid-phase peptide synthetic techniques and subsequent improvements (e.g., Merrifield, 1963 J. Am. Chem. Soc. 85:2149; Mitchell et al., 1978 J. Org. Chem. 43:2485; Albericio, F. (2000). Solid- Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press; Nilsson et al., 2005 Annu. Rev. Biophys. Biomol. Struct. 34; Schnolzer et al., Int. J. Peptide Res. Therap. 13 (1–2): 31; Li et al. 2013 Molecules 18:9797) are routine in the peptide synthesis art and may be employed to chemically synthesize the herein described peptides.

Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be fused in-frame or conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. Fusion domain polypeptides may be joined to the polypeptide at the N-terminus and/or at the C-terminus, and may include as non-limiting examples, immunoglobulin-derived sequences such as Ig constant region sequences or portions thereof, affinity tags such as His tag (e.g., hexahistidine or other polyhistidine), FLAG™ or myc or other peptide affinity tags, detectable polypeptide moieties such as green fluorescent protein (GFP) or variants thereof (e.g., yellow fluorescent protein (YFP), blue fluorescent protein (BFP), other aequorins or derivatives thereof, etc.) or other detectable polypeptide fusion domains, enzymes or portions thereof such as glutathione-S- transferase (GST) or other known enzymatic detection and/or reporter fusion domains, and the like, as will be familiar to the skilled artisan.

Recombinant protein expression systems are known in the art and may in certain embodiments be used to produce the herein described peptides. For example, certain bacterial expression systems such as E. coli recombinant protein expression systems yield polypeptide products having N-terminal formylated methionine. In some situations the peptide may therefore comprise an N-terminal methionine residue (which may be unmodified methionine or formyl-methionine or another methionine analog, variant, mimetic or derivative as provided herein), sometimes referred to as initiator methionine, immediately preceding the peptide sequence. Also contemplated are embodiments in which one or more of the herein described peptides containing N- terminal methionine (e.g., as methionine or N-formylmethionine) may be recombinantly expressed according to art-accepted practices in a host cell that also expresses methionine aminopeptidase (MAP), an enzyme that is capable of cleaving the N-terminal methionine to remove it from the nascent polypeptide product. See, e.g., Natarajan et al., 2011 PLoS ONE 6(5): e20176; Shen et al., 1993 Proc. Natl. Acad. Sci. USA 90:8108; Shen et al., 1997 Prot. Eng. 10:1085. Alternatively, the MAP enzyme itself may be produced recombinantly (e.g., Tsunasawa et al., 1997 J. Biochem. 122:843; Bradshaw et al., 1998 Trends Bioch. Sci. 23:263; Ben-Bassat et al., 1987 J. Bacteriol. 169:751) or obtained commercially (Sigma-Aldrich, St. Louis, MO, e.g., catalog number M6435) and used to remove N-terminal methionine from the present peptides post-synthesis.

According to certain preferred embodiments a peptide for inclusion in the herein disclosed conjugate may comprise a peptide, polypepetide or peptidomimetic that includes, or that shares close sequence identity to or structural features with, a polypeptide of at least 6 and no more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7 or 6 amino acids, for example a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOS:1-10, wherein the conjugate in which the peptide is present has membrane permeabilization capability (e.g., as an AMP or CPP) that can be detected according to established criteria (e.g., Mishra et al, Proc. Natl. Acad. Sci. U. S. A.108, 16883-16888 (2011); Schmidt et al, J. Am. Chem. Soc. 133, 6720-6727 (2011); Schmidt et al, J. Am. Chem. Soc. 134, 19207-19216 (2012); Hu et al, Macromolecules 46, 1908-1915 (2013), and references cited therein). As described above, in certain preferred embodiments the peptide for inclusion in the herein disclosed conjugate may comprise a peptide, polypepetide or peptidomimetic that includes, or that shares close sequence identity to a peptide comprising the amino acid sequence set forth in any one of SEQ ID NOS:1-10, wherein the conjugate in which the peptide is present has membrane permeabilization capability (e.g., as an AMP or CPP) that can be detected according to established criteria. Methods for the determination of aminoglycoside antibiotic activity via specific targeting to ribosomes are also known in the art. For example, characterization of the ribosomal binding site of aminoglycosides is described in Vicens et al., 2002 Chem. Biol.9:747-755; and Fourmyet al., 1996 Science 274:1367; methodologies for the measurement of aminoglycoside binding to ribosomes are disclosed, for example, in Chang and Flaks, 1972 Antimicrob. Agents Chemother. 2:294-307; Boeck et al., 1979 FEBS Lett. 104:317-321; and Llano-Sotelo et al. 2009 RNA 15:1597-1604 (2009). Assay methods for determining antibacterial activity of aminoglycosides are known in the art.

As generally referred to in the art, and as used herein, sequence identity and sequence homology may be used interchangeably and generally refer to the percentage of nucleotides or amino acid residues in a candidate sequence that are identical with, respectively, the nucleotides or amino acid residues in a reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and optionally not considering any conservative substitutions as part of the sequence identity. In certain embodiments, a peptide such as an AMP or CPP peptide of the embodiments disclosed herein shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, 91%, 92%, 93% or 94%, or at least about 95%, 96%, 97%, 98%, or 99% of the amino acid residues (or of the nucleotides in a polynucleotide encoding such a peptide) with the sequence of a general formula according to any one of SEQ ID NOS:1-10.

Such sequence identity may be determined according to well known sequence analysis algorithms, including those available from the University of Wisconsin Genetics Computer Group (Madison, WI), such as FASTA, Gap, Bestfit, BLAST, or others.

"Natural or non-natural amino acid" includes any of the common naturally occurring amino acids which serve as building blocks for the biosynthesis of peptides, polypeptides and proteins (e.g., alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), tyrosine(Y)) and also includes modified, derivatized, enantiomeric, rare and/or unusual amino acids, whether naturally occurring or synthetic, for instance, N-formylmethionine, hydroxyproline, hydroxylysine, desmosine, isodesmosine, ^-N-methyllysine, ^-N-trimethyllysine, methylhistidine, dehydrobutyrine, dehydroalanine, ^-aminobutyric acid, ^-alanine, ^- aminobutyric acid, homocysteine, homoserine, citrulline, ornithine and other amino acids that may be isolated from a natural source and/or that may be chemically synthesized, for instance, as may be found in Proteins, Peptides and Amino Acids Sourcebook (White, J.S. and White, D.C., 2002 Humana Press, Totowa, NJ) or in Amino Acid and Peptide Synthesis (Jones, J., 2002 Oxford Univ. Press USA, New York) or in Unnatural Amino Acids, ChemFiles Vol. 1, No. 5 (2001 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, MO) or in Unnatural Amino Acids II, ChemFiles Vol.2, No.4 (2002 Fluka Chemie GmbH; Sigma-Aldrich, St. Louis, MO). Additional descriptions of natural and/or non-natural amino acids may be found, for example, in Kotha, 2003 Acc. Chem. Res.36:342; Maruoka et al., 2004 Proc. Nat. Acad. Sci. USA 101:5824; Lundquist et al., 2001 Org. Lett. 3:781; Tang et al., 2002 J. Org. Chem. 67:7819; Rothman et al., 2003 J. Org. Chem. 68:6795; Krebs et al., 2004 Chemistry 10:544; Goodman et al., 2001 Biopolymers 60:229; Sabat et al., 2000 Org. Lett. 2:1089; Fu et al., 2001 J. Org. Chem. 66:7118; and Hruby et al., 1994 Meths. Mol. Biol.35:249. The standard three-letter abbreviations and one-letter symbols are used herein to designate natural and non-natural amino acids.

Other non-natural amino acids or amino acid analogues are known in the art and include, but are not limited to, non-natural L or D derivatives (such as D-amino acids present in peptides and/or peptidomimetics such as those presented above and elsewhere herein), fluorescent labeled amino acids, as well as specific examples including O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl- phenylalanine, 3-idio-tyrosine, O-propargyl-tyrosine, homoglutamine, an O-4-allyl-L- tyrosine, a 4-propyl-L-tyrosine, a 3-nitro-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L- phenylalanine, a p-acyl-L-phenylalanine, a p-acetyl-L-phenylalanine, an m-acetyl-L- phenylalanine, selenomethionine, telluromethionine, selenocysteine, an alkyne phenylalanine, an O-allyl-L-tyrosine, an O-(2-propynyl)-L-tyrosine, a p- ethylthiocarbonyl-L-phenylalanine, a p-(3-oxobutanoyl)-L-phenylalanine, a p- benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, homoproparglyglycine, azidohomoalanine, a p-iodo- phenylalanine, a p-bromo-L-phenylalanine, dihydroxy-phenylalanine, dihydroxyl-L- phenylalanine, a p-nitro-L-phenylalanine, an m-methoxy-L-phenylalanine, a p-iodo- phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, and an isopropyl-L-phenylalanine, trifluoroleucine, norleucine (“Nle”), D-norleucine (“dNle” or “D-Nle”), 5-fluoro-tryptophan, para-halo-phenylalanine, homo-phenylalanine (“homo-Phe”), seleno-methionine, ethionine, S-nitroso-homocysteine, thia-proline, 3- thienyl-alanine, homo-allyl-glycine, trifluoroisoleucine, trans and cis-2-amino-4- hexenoic acid, 2-butynyl-glycine, allyl-glycine, para-azido-phenylalanine, para- cyano-phenylalanine, para-ethynyl-phenylalanine, hexafluoroleucine, 1,2,4-triazole- 3-alanine, 2-fluoro-histidine, L-methyl histidine, 3-methyl-L-histidine, β-2-thienyl-L- alanine, β-(2-thiazolyl)-DL-alanine, homoproparglyglycine (HPG) and azidohomoalanine (AHA) and the like.

In certain embodiments a natural or non-natural amino acid may be present that comprises an aromatic side chain, as found, for example, in phenylalanine or tryptophan or analogues thereof including in other natural or non-natural amino acids based on the structures of which the skilled person will readily recognize when an aromatic ring system is present, typically in the form of an aromatic monocyclic or multicyclic hydrocarbon ring system consisting only of hydrogen and carbon and containing from 6 to 19 carbon atoms, where the ring system may be partially or fully saturated, and which may be present as a group that includes, but need not be limited to, groups such as fluorenyl, phenyl and naphthyl.

In certain embodiments a natural or non-natural amino acid may be present that comprises a hydrophobic side chain as found, for example, in alanine, valine, isoleucine, leucine, proline, phenylalanine, tryptophan or methionine or analogues thereof including in other natural or non-natural amino acids based on the structures of which the skilled person will readily recognize when a hydrophobic side chain (e.g., typically one that is non-polar when in a physiological milieu) is present. In certain embodiments a natural or non-natural amino acid may be present that comprises a basic side chain as found, for example, in lysine, arginine or histidine or analogues thereof including in other natural or non-natural amino acids based on the structures of which the skilled person will readily recognize when a basic (e.g., typically polar and having a positive charge when in a physiological milieu) is present.

Peptides disclosed herein may in certain embodiments include L- and/or D- amino acids so long as the biological activity of the peptide is maintained (e.g., membrane-disruptive antimicrobial peptide (AMP) and/or cell penetrating peptide (CPP) activity). The peptides also may comprise in certain embodiments any of a variety of known natural and artificial post-translational or post-synthetic covalent chemical modifications by reactions that may include glycosylation (e.g., N-linked oligosaccharide addition at asparagine residues, O-linked oligosaccharide addition at serine or threonine residues, glycation, or the like), fatty acylation, acetylation, formylation, PEGylation, and phosphorylation. Peptides herein disclosed may further include analogs, alleles and allelic variants which may contain amino acid deletions, or additions or substitutions of one or more amino acid residues with other naturally occurring amino acid residues or non-natural amino acid residues.

Peptide and non-peptide analogs may be referred to as peptide mimetics or peptidomimetics, and are known in the pharmaceutical industry (Fauchere, J. Adv. Drug Res. 15:29 (1986); Evans et al. J. Med. Chem. 30: 1229 (1987)). These compounds may contain one or more non-natural amino acid residue(s), one or more chemical modification moieties (for example, glycosylation, pegylation, fluorescence, radioactivity, or other moiety), and/or one or more non-natural peptide bond(s) (for example, a reduced peptide bond: --CH 2 -NH 2 --). Peptidomimetics may be developed by a variety of methods, including by computerized molecular modeling, random or site-directed mutagenesis, PCR-based strategies, chemical mutagenesis, and others.

As also described above, certain embodiments also relate to peptidomimetics, or“artificial” polypeptides. Such polypeptides may contain one or more amino acid insertions, deletions or substitutions, one or more altered or artificial peptide bond, one or more chemical moiety (such as polyethylene glycol, glycosylation, label, toxin, or other moiety), and/or one or more non-natural amino acid. Synthesis of peptidomimetics is well known in the art and may include altering naturally occurring proteins or polypeptides by chemical mutagenesis, single or multi- site-directed mutagenesis, PCR shuffling, use of altered aminoacyl tRNA or aminoacyl tRNA synthetase molecules, the use of“stop” codons such as amber suppressors, use of four or five base-pair codons, or other means. Linker Moiety

A linker moiety refers to a diverse group of covalent linkages that couples the aminoglycoside to the peptide. The covalent linkage can be a combination of stable chemical bonds, optionally including single, double, triple or aromatic carbon-carbon bonds, as well as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogen bonds.

A linker moiety is typically derived from one or more linker compounds that are bi-functional. A bi-functional linker compound comprises two terminal functional groups, one of which for binding to the aminoglycoside portion (which may be an aminoglycoside or an aminoglycoside derivatized to contain a functionality for coupling with the linker compound), the other for binding to the peptide portion (which may be a peptide or a peptide derivatized to contain a functionality for coupling with the linker compound). The binding typically results in the formation of stable covalent bonds, such as carboxylate ester, ether, thiol ether or amide moieties.

Typically, a linker moiety has 1-40 non-hydrogen atoms selected from the group consisting of C, N, O, P, and S; and are composed of any combination of ether, amide, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic, heteroaromatic, heterocyclic bonds. The covalent linkage can be linear or cyclic, or a combination thereof. Examples of linear linkages include polymethylene, polyethyleneglycol, alkylsulfonyl, alkylthio, amino alcohol and diol. “Amino alcohol” refers to a diradical of the formula–NH-Y-O-, wherein Y is an alkanediyl or substituted alkanediyl, alkanediyl refers to a divalent alkyl with two hydrogen atoms taken from different carbon atoms. “Diol” refers to a diradical of the formula– O-Y-O-, wherein Y is defined as above. Formation of the Conjugates

Once the aminoglycoside and the peptide are selected according to the above design rules, they can be constructed such that the cationic and hydrophobic contributions of the peptide portion together with those from the aminoglycoside portion create a composite molecule (i.e., a P-L-A conjugate) which lies on the global trendline set by AMP or CPP saddle-splay curvature selection rules. By itself, the amino glycoside does not satisfy the saddle-splay curvature design rules, so it will not independently display selective membrane permeabilization activity. By itself, the peptide may or may not satisfy the saddle-splay curvature design rules, such that it may not independently display selective membrane permeabilization activity.

As discussed above, the aminoglycoside and the peptide are conjugated together via a linker moiety. In certain embodiments, the linker moiety may be coupled to the peptide by an amide bond formation either at the C-terminal or at the N-terminal, and to the aminoglycoside portion by a forming a bond with a functional group thereof, including, without limitation, a primary or second hydroxyl group, a primary or secondary amino group, and the like. The resulting linkage may be, for example, an ester, an amide, an ether, a thioether, an amine, etc.

In various embodiments, the coupling reactions include known methods in the art, such as amide bond formation (e.g., via succinimide coupling), reductive amination,“click” chemistry, thiol-maleimide coupling at either the N-terminal or C- terminal ends of the peptide after modification with appropriate functionality of both, the peptide and the aminoglycoside. Coupling can take place either in solution or solid phase. Scheme 1 shows a number of approaches that result in covalently coupling the peptide and the aminoglycoside portions by a linker moiety.

Scheme 2 shows a synthetic pathway for coupling an exemplary aminoglycoside (tobramycin) and a 12 amino acid peptide (RQIKIWFQNRRW, SEQ ID NO:9), the resulting conjugate is referred to herein as Pentobra (or pentobra).

a) Boc2O, TEA, H2O/DMF (1:4), 5 h, 60°C, 93%; b) succinic anhydride, DMAP, pyridine, 4 days, 23°C, 69%; c) fully protected RQIKIWFQNRRW [SEQ ID NO:9] (compound 3), DIEA, HBTU, HOBt, DMF, 24 h, 23°C; d) TFA/phenol/water/thioanisole/TIS (10:0.7:0.5:0.5:0.25 v:w:v:v:v), 3 h, 23°C, 49%.

Using tobramycin and the 12-AA peptide (RQIKIWFQNRRW [SEQ ID NO:9]) as examples, the following schemes (Schemes 3-6) illustrate alternative synthetic approaches for linking an aminoglyscoside to a peptide: Scheme 3– amide coupling to C-terminal

Scheme 4– thiol-maleimide coupling to N-terminal

Additional conjugates of the 12-AA peptide (RQIKIWFQNRRW [SEQ ID NO:9]) with other aminoglycosides can be formed by similar approaches as illustrated in Schemes 1-6. In various embodiments, the conjugate may be (Pen-L- aminoglycoside):

The conjugates described herein may generally be used as the free base. Alternatively, the compounds may be used in the form of acid addition salts. Acid addition salts of the free base amino compounds may be prepared according to methods well known in the art, and may be formed from organic and inorganic acids. Suitable organic acids include (but are not limited to) maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acids include (but are not limited to) hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Thus, the term “pharmaceutically acceptable salt” as well as any and all specific conjugates described herein is intended to encompass any and all pharmaceutically suitable salt forms.

Furthermore, some of the crystalline forms of any compound described herein, including the salt form, may exist as polymorphs. In addition, some of the compounds may form solvates with water or other organic solvents. Often crystallizations produce a solvate of the disclosed compounds. As used herein, the term“solvate” refers to an aggregate that comprises one or more molecules of any of the disclosed compounds with one or more molecules of solvent. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the presently disclosed compounds may exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, as well as the corresponding solvated forms. Adjuvants

In certain embodiments disclosed herein for the first time, compositions are provided that comprise the presently described multifunctional membrane-active aminoglycoside peptide (P-L-A) conjugate and that further comprise an adjuvant moiety as described herein. Preferably and in certain embodiments the adjuvant moiety comprises at least one saccharide compound or one glycomimetic adjuvant moiety as provided herein.

The adjuvant moiety may in certain embodiments be provided with the conjugate in a form that permits the conjugate and the adjuvant to be administered to a subject or contacted with target bacteria simultaneously or sequentially and in either order. In certain other embodiments, the adjuvant may be provided in combination with, in a mixture with, in non-covalent association with, or covalently linked to the conjugate.

Covalent linkage of the adjuvant moiety to the conjugate may be achieved through chemical modifications such as those described herein for conjugate formation, or by other conventional chemical steps with which those skilled in the art will be familiar, depending on the type and location of appropriate chemically reactive functionalities that may be present in the structures of the conjugate and the adjuvant, or that may be introduced to the conjugate and/or the adjuvant by way of no more than routine chemical methodologies. The adjuvant moiety, whether a saccharide compound as provided herein or a glycomimetic adjuvant moiety as provided herein, may be covalently linked to the P-L-A conjugate via attachment sites on any of the peptide, linker or aminoglycoside portions of the conjugate. Certain embodiments contemplate covalent attachment of the adjuvant moiety to the P-L-A conjugate via a chemical bond formed between the adjuvant moiety and the peptide moiety, such as via a carboxy-terminus of the peptide, or via an amino terminus of the peptide, or via an available chemical group that forms part of an amino acid side chain in the peptide. Non-limiting examples of chemical linkages between the adjuvant moiety and the peptide of the P-L-A conjugate include ester, ether, disulfide, and amide linkages, although other standard conjugation linkages may also be employed as will be known to those familiar with the art.

Hence, in certain embodiments the adjuvant may be covalently linked to the peptide moiety of the conjugate; in certain embodiments the adjuvant may be covalently linked to the aminoglycoside moiety of the conjugate; and in certain embodiments the adjuvant may be covalently linked to the linker moiety of the conjugate. It will be appreciated that when an adjuvant is covalently linked to the conjugate, the composite conjugate-adjuvant molecule may in certain embodiments still be generated with a chemical structure that adheres to the herein disclosed design principles, such as having a peptide P that exhibits the structural and physicochemical properties as described elsewhere herein, having a total charge Q T of from 3 + to 10 + as described herein, and/or wherein the conjugate-adjuvant molecule has a composite octanol:water partition coefficient of from -1.5 to zero as described herein.

In certain embodiments the adjuvant moiety may comprise a saccharide compound, which as used herein may include, for example by way of illustration but not limitation, any proton-motive force-stimulating compound disclosed in WO 2012/151474 or in Allison et al. (2011 Nature 473:216). Examples of saccharide compounds that may be proton-motive force-stimulating compounds include glucose, mannitol, fructose and pyruvate. Other examples of proton-motive force-stimulating compounds contemplated herein as “saccharide compounds” include lactate, glucosamine, sorbitol, N-acetyl glucosamine, N-acetyl muramic acid, maltose, galactosamine, trehaloseribose, arabinose, gluconate, glycerol, galactarate, and analogs or derivative thereof.

According to non-limiting theory, however, the present aminoglycoside compositions operate according to a different mechanism that relates to the herein- described deliberately designed membrane-disruptive properties of the peptide P, which does not rely on PMF to gain entry into cells and thus could not, from Allison et al. (2011), have been predicted to synergize with an adjuvant moiety such as a saccharide compound or a glycomimetic adjuvant moiety as presently disclosed. In particular, where the peptide P of the presently disclosed P-L-A conjugates has physicochemical properties (e.g., significantly higher molecular weight that is typically greater than two-, three-, or four-fold that of tobramycin; and substantially different charge, hydrophobicity, and solubility) that differ substantially from the properties of tobramycin as disclosed by Allison et al., the present aminoglycoside compositions, which comprise an adjuvant moiety and a P-L-A conjugate having a relatively bulky, amphipathic, and membrane-disruptive peptide (P), are not believed to depend entirely (if at all) on PMF stimulation to promote cellular uptake, as is the case in Allison et al.

Adjuvant moieties that are saccharide compounds as provided herein may in certain embodiments be covalently linked to the herein described P-L-A conjugates via standard chemical conjugation techniques, including, for example, those described elsewhere herein. Representative linkages for such aminoglycoside compositions include esters, ethers, thioethers, disulfides, amines and/or amides. For example, the saccharide compound may be conjugated to the peptide moiety of the herein described P-L-A conjugate according to established methodologies, through a terminal amine or carboxylic acid, or through reaction with a modified N- or C- terminus on the peptide, or through another reaction with an amino acid within the peptide chain that comprises the peptide P.

In certain embodiments the adjuvant moiety may comprise a glycomimetic adjuvant moiety. By way of a brief background, oligo- and polysaccharides fulfill a myriad of important roles in human biology and in human health. Because of the many reactive functional groups on each saccharide monomer, however, the synthesis of even small oligosaccharides with well-defined structures is very tedious, and very few techniques exist to produce well-defined oligo- and polysaccharides in substantial quantities. As an alternative, monomers containing a saccharide group can be polymerized to produce polymers generally referred to as glycomimetic polymers. Polymeric glycomimetic products may be comprised of a small number of repeating monosaccharide, disaccharide, or trisaccharide subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats) but high molecular weight (e.g., up to about 3,000 Da, 5,000 Da, 7,500 Da, 9,000 Da or 10,000– 15,000 Da) glycomimetic polymers can also be achieved.

Glycomimetic polymers are therefore known in the art, but most reported glycomimetics are linear and typically contain one or two types of saccharide residues. Thus, although there is a large body of work describing the synthesis of glycomimetic polymers, nearly all of the reported approaches fail to recapitulate two key features of natural oligo- and polysaccharides: branching, and incorporation of sugar subunits into the polymer backbone in their ring-closed form. Lin and Kasko ((2013 Biomacromolecules 14(2):350) describe a technique that recaptures both of these structural features, and demonstrate that both features enhance the activity of glycomimetic polymer-lectin interactions, in particular, specific binding to human mannose-binding lectin.

Glycomimetic adjuvant moieties for use according to certain of the presently contemplated embodiments may, however, include linear and branched oligomers and polymers, in which the repeating subunit is comprised of a polymerizable group that incorporates a monosaccharide, disaccharide, trisaccharide or other oligosaccharide residue. The polymerizable group may comprise an unsaturated bond that is polymerized using standard polymerization techniques known in the art, or may include reactive units that polymerize via condensation polymerization, ring opening polymerization or other step-growth mechanisms. Exemplary repeating subunits may thus comprise, according to certain presently disclosed embodiments, polymerizable groups that may be one or more repeating units selected from poly(vinyl alcohol), poly(hydroxylethylmethacrylates), poly(hydroxyethylacrylates), poly(ester)s, poly ^- hydroxyesters, proteins, poly(oxazoline), polyamino acids, poly(lactides), poly(styrenes), poly(acrylates), poly(methacrylates), poly(vinylethers), polyethylenes, and poly(ethylene imine)s. The repeating subunit preferably further comprises pendant side chains capable of condensing with a monosaccharide, a disaccharide, and/or a trisaccharide, to provide linear and/or branched polymers that may be formed from a plurality of repeating subunits, which subunits may comprise mono-, di- and/or trisaccharides.

Accordingly, in certain presently disclosed embodiments there is provided an aminoglycoside composition that comprises (1) a multifunctional membrane-active aminoglycoside peptide conjugate (P-L-A) as described herein; and (2) at least one glycomimetic adjuvant moiety that is selected from: (a) a linear homopolymer formed from a monosaccharide repeating subunit, (b) a linear copolymer formed from at least two different monosaccharide repeating subunits, (c) a branched homopolymer formed from a monosaccharide repeating subunit, and (d) a branched homopolymer formed from at least two different monosaccharide repeating subunits. In certain further embodiments the glycomimetic adjuvant moiety comprises a branched polymer formed from at least one monosaccharide repeating subunit wherein one or a plurality of branch points in the branched polymer comprise the monosaccharide in closed-ring form. Examples of such glycomimetics are described, for instance, in Lin et al. (2013 Biomacromolec.14(2):350).

For example, a polymer of five to about 250 repeating subunits may be formed from a polymeric backbone structure of repeating subunits having pendant side chains capable of condensation reactions with saccharide compounds or with monosaccharides, disaccharides, and/or trisaccharides, wherein the backbone structure comprises one or more repeating subunits selected from poly(vinyl alcohol), poly(hydroxylethylmethacrylates), poly(hydroxyethylacrylates), poly(ester)s, poly ^- hydroxyesters, proteins, poly(oxazoline), polyamino acids, poly(lactides), poly(styrenes), poly(acrylates), poly(methacrylates), poly(vinylethers), polyethylenes, and poly(ethylene imine)s.

Polymers of five 5 to about 250 repeating subunits may be formed by reacting polymeric subunits having reactive end groups and/or side chains with saccharide compounds or with monosaccharides, disaccharides, and/or trisaccharides, wherein the reactive end group may be selected from acrylate, methacrylate, styrene, allyl ether, vinyl ether, epoxide, cyanoacrylate, isocyanate, triazide, phosphazine, imine, ethylene imine, oxazoline, propylene sulfides, groups polymerizable via condensation reactions, alkene, alkyne,“click” chemistry reactive groups, and carboxylic acid. The repeating subunits may be polymerized directly, or monosaccharides, disaccharides, and/or trisaccharides may be attached to a polymer backbone directly, and/or attached to a polymer backbone through a spacer, according to chemical approaches disclosed herein and known in the art, such as the above described linker chemistries. Accordingly, in certain embodiments the present glycomimetic adjuvant moiety may be produced by polymerizing reactive monomers, or by post-polymerization modification of a polymer through condensation reactions, click chemistry, or other standard conjugation techniques.

For instance, glycomimetic adjuvant moieties may comprise glycomimetic polymers that can be produced by the polymerization of monomers containing mono-, di- and/or trisaccharide residues, to arrive at polymers of repeating subunits. Common techniques for glycomimetic polymer preparation are known in the art and may include, by way of non-limiting example, atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), free radical polymerization, ring opening polymerization, and polycondensation reactions. Branching may be introduced through the incorporation of multifunctional groups such as agents having two polymerizable groups, a polymerizable group and a chain transfer group, or molecules with multiple reactive sites for condensation reactions. General methodologies for the synthesis of linear and branched glycopolymers are described, for example, in Lin and Kasko (2013, Biomacromolecules 14(2):350) and Liau et al. (2015 Biomacromolecules 16(1):284). MONOMERS. Exemplary monomer structures are contemplated for use in repeating subunits with which to form a glycomimetic adjuvant moiety according to certain presently disclosed embodiments, and include structural formulae [I] and [II] below.

Accordingly in certain embodiments the glycomimetic adjuvant moiety may comprise a molecule of structural formula [I] (above) or its stereoisomers, in which one or more R groups (R1-R5) is a reactive end group selected from acrylate, methacrylate, styrene, allyl ether, vinyl ether, epoxide, cyanoacrylate, isocyanate, triazide, phosphazine, imine, ethylene imine, oxazoline, propylene sulfides, groups polymerizable via condensation reactions, alkene, alkyne,“click” chemistry, and carboxylic acid.

In certain other embodiments the glycomimetic adjuvant moiety may comprise a molecule of structural formula [I] (above) or its stereoisomers, in which one or more R groups (R2-R5) and/or Y contains a reactive end group selected from acrylate, methacrylate, styrene, allyl ether, vinyl ether, epoxide, cyanoacrylate, isocyanate, triazide, phosphazine, imine, ethylene imine, oxazoline, propylene sulfides, groups polymerizable via condensation reactions, alkene, alkyne,“click” chemistry, and carboxylic acid. In structural formulae [I] and [II] one or more of the X groups may be hydrogen; or one or more of the X groups may be a functional group selected from amine, amide, alcohol, sulfate, carboxylic acid or other functional group. POLYMER ASSEMBLY. Glycomimetic polymers may be synthesized by the polymerization according to standard methodologies of monomeric subunits containing mono-, di- or trisaccharide moieties. Common techniques for such synthesis may include ATRP, RAFT, free radical polymerization, ring opening polymerization, and polycondensation reactions. Branching may be introduced through the incorporation of multifunctional groups such as agents having two polymerizable groups, a polymerizable group and a chain transfer group, or molecules with multiple reactive sites for condensation reactions. A representative example is shown in Scheme 7:

Scheme 7. Synthesis of poly(glucose) [See Example 8.]

COVALENT LINKAGE OF GLYCOMIMETIC POLYMER TO P-L-A CONJUGATE. The synthetic glycomimetic polymer prepared for use as a glycomimetic adjuvant moiety in certain embodiments of the present aminoglycoside composition may be conjugated to the P-L-A conjugate through terminal amine or carboxylic acid of the peptide (P), or through reaction with a modified N- or C- terminus of the peptide, or through reaction with an amino acid within the peptide chain, using standard conjugation techniques. By way of non-limiting example, two approaches for such conjugation are described here.

(a) A glycomimetic polymer such as polyglucose prepared according to Scheme 7 may have a thiol group at the polymer chain end that can be used for attaching the glycomimetic polymer to the peptide of a P-L-A conjugate constructed to have a free carboxy terminus. Briefly, during the peptide synthesis of the peptide P, a cysteine residue may be added to the C-terminus to yield N-peptide-Cys-C; the C-terminal cysteine can be covalently bonded to the polyglucose polymer through disulfide bridge formation between the thiol of the cysteine residue and the terminal thiol of polyglucose (Scheme 8, shown for Pentobra).

Scheme 8. Conjugation of polyglucose to CysPentobra (pentobra with an added C- terminal cysteine residue) through disulfide bond. (b) Alternatively, the terminal thiol group of the glycomimetic polymer may be modified with 2-maleimidoethylamine to yield a free amine group that can react with the C-terminus of the Pentobra peptide (P) via amide bond formation (Scheme 9). The coupling requires fully protecting all of the amine groups of Pentobra, for example, by cleaving the peptide moiety P from the resin on which it is synthesized without deprotection of its side chains.

Scheme 9. Conjugation of modified polyglucose to protected Pentobra through amide bond. Polyglucose is previously modified with 2-maleimidoethylamine. The coupling is performed in the presence of N,N-diisopropylethylamine (DIEA) and coupling agents (HOBt and HBTU). The side chains are finally deprotected using trifluoroacetic acid (TFA). Pharmaceutical Compositions and Administration

The present invention also relates in certain embodiments to pharmaceutical compositions containing the conjugates of the invention disclosed herein, including pharmaceutical compositions that comprise the herein-described aminoglycoside composition which comprises a multifunctional membrane-active aminoglycoside peptide conjugate (P-L-A) as described herein and an adjuvant moiety, such as a saccharide compound and/or a glycomimetic adjuvant moiety. In one embodiment, the present invention relates to a pharmaceutical composition comprising the aminoglycoside-peptide conjugate (and optionally further comprising an adjuvant moiety) in a pharmaceutically acceptable excipient, carrier or diluent and in an amount effective to confer antimicrobial benefit for a condition that would benefit from a decreased level of bacteria, such as a bacterial infection, when administered to an animal, preferably a mammal, most preferably a human.

Administration of the compounds disclosed herein, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions can be prepared by combining a herein disclosed compound with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, intranasal, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, subcutaneous, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, intraocular, via local delivery, subcutaneous, intraadiposal, intraarticularly or intrathecally. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical compositions are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein disclosed compound in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain a therapeutically effective amount of a herein disclosed compound, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings of this disclosure. Embodiments of the invention include compositions that may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.

The pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids, such as water, saline, glycerol and ethanol, and the like. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).

A pharmaceutical composition according to certain embodiments of the invention may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel™, corn starch and the like; lubricants such as magnesium stearate or Sterotex™; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition intended for either parenteral or oral administration should according to certain embodiments contain an amount of a herein disclosed compound such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a herein disclosed compound in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Preferred oral pharmaceutical compositions contain between about 4% and about 50% of the compound. Preferred pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the compound prior to dilution.

The pharmaceutical composition may be intended for topical administration (e.g., for treating acne, including acne vulgaris), in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of a herein disclosed compound of from about 0.1 to about 10% w/v (weight per unit volume).

The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule.

The pharmaceutical composition in solid or liquid form may include an agent that binds to a herein disclosed compound and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome.

The pharmaceutical composition may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of the herein disclosed compounds may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols.

The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a herein disclosed compound with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.

The compounds according to certain embodiments of the invention disclosed herein, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose is (for a 70 Kg mammal) from about 0.001 mg/Kg (i.e., 0.07 mg) to about 100 mg/Kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 Kg mammal) from about 0.01 mg/Kg (i.e., 0.7 mg) to about 50 mg/Kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 Kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/Kg (i.e., 1.75 g).

The ranges of effective doses provided herein are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts. (see, e.g., Berkowet al., eds., The Merck Manual, 16 th edition, Merck and Co., Rahway, N.J., 1992; Goodman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., eds., Remington's Pharmaceutical Sciences, 18 th edition, Mack Publishing Co., Easton, PA (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, CT (1992)).

The total dose required for each treatment can be administered by multiple doses or in a single dose over the course of the day, if desired. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The diagnostic pharmaceutical compound or composition can be administered alone or in conjunction with other diagnostics and/or pharmaceuticals directed to the pathology, or directed to other symptoms of the pathology. The recipients of administration of the herein disclosed compounds and/or compositions can be any vertebrate animal, such as mammals. Among mammals, the preferred recipients are mammals of the Orders Primate (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs). Among birds, the preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipients are humans.

For topical applications, it is preferred to administer an effective amount of a pharmaceutical composition to a target area, e.g., skin surfaces, mucous membranes, and the like. This amount will generally range from about 0.0001 mg to about 1 g of a herein disclosed compound per application, depending upon the area to be treated, whether the use is diagnostic, prophylactic or therapeutic, the severity of the symptoms, and the nature of the topical vehicle employed. A preferred topical preparation is an ointment, wherein about 0.001 to about 50 mg of active ingredient is used per cc of ointment base. The pharmaceutical composition can be formulated as transdermal compositions or transdermal delivery devices ("patches"). Such compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive. Such transdermal patches may be used to provide continuous pulsatile, or on demand delivery of the herein disclosed compounds as desired.

The pharmaceutical compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U.S. Pat. Nos.3,845,770 and 4,326,525 and in P. J. Kuzma et al., Regional Anesthesia 22 (6): 543-551 (1997), all of which are incorporated herein by reference.

The compositions can also be delivered through intra-nasal drug delivery systems for local, systemic, and nose-to-brain medical therapies. Controlled Particle Dispersion (CPD)™ technology, traditional nasal spray bottles, inhalers or nebulizers are known by those skilled in the art to provide effective local and systemic delivery of drugs by targeting the olfactory region and paranasal sinuses.

The invention also relates in certain embodiments to an intravaginal shell or core drug delivery device suitable for administration to the human or animal female. The device may be comprised of the active pharmaceutical ingredient in a polymer matrix, surrounded by a sheath, and capable of releasing the compound in a substantially zero order pattern on a daily basis similar to devices used to apply testosterone as described in PCT Published Patent No. WO 98/50016.

Current methods for ocular delivery include topical administration (eye drops), subconjunctival injections, periocular injections, intravitreal injections, surgical implants and iontophoresis (uses a small electrical current to transport ionized drugs into and through body tissues). Those skilled in the art would combine the best suited excipients with the compound for safe and effective intra-ocular administration.

The most suitable route will depend on the nature and severity of the condition being treated. Those skilled in the art are also familiar with determining administration methods (e.g. oral, intravenous, inhalation, sub-cutaneous, rectal, etc.), dosage forms, suitable pharmaceutical excipients and other matters relevant to the delivery of the compounds to a subject in need thereof.

As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 5%, 6%, 7%, 8% or 9%. In other embodiments, the terms“about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%, 11%, 12%, 13% or 14%. In yet other embodiments, the terms “about” or“approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 16%, 17%, 18%, 19% or 20%. Method of Treatment

High-resolution synchrotron x-ray scattering shows that the tobramycin - peptide conjugate (Pentobra), but not tobramycin, can generate negative Gaussian curvature in model bacteria cell membranes, which is topologically required for membrane permeation mechanisms, such as pore formation, budding, and blebbing. The X-ray data are consistent with bacterial inner membrane permeability results from an E. coli ML35 reporter strain. Plate killing assays demonstrate the advantage of imparting tobramycin with membrane activity as Pentobra is able to maintain robust bactericidal activity against E. coli and S. aureus persister cells, whereas tobramycin was not active. These results demonstrate that membrane curvature design rules can deterministically inform the construction of multifunctional antibiotics, and thereby broaden the spectrum of activity of single target drugs to bacterial sub-populations like persisters.

Thus, certain embodiments relate to a method of treating a bacterial infection in a mammal, including infection by antibiotic-resistant bacteria, comprising administering to the mammal a therapeutically effective amount of an aminoglycoside-peptide conjugate (P-L-A), as described herein.

"Mammal" includes humans, and also includes domesticated animals such as laboratory animals, livestock and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and also includes non-domesticated animals such as wildlife and the like.

“Therapeutically effective amount” refers to that amount of a compound or conjugate of the disclosure which, when administered to a mammal, preferably a human, is sufficient to effect treatment, as defined below, of a disease or condition in the mammal, preferably a human. The amount of a compound of the disclosure which constitutes a“therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

“Treating” or“treatment” as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or disorder of interest (e.g., bacterial infection), and includes:

(i) inhibiting or preventing growth, reproduction or any metabolic activity of target organisms, killing or injuring the same, to a substantial and statistically significant degree of inhibition (though not necessarily complete), e.g., at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or greater inhibition relative to appropriate untreated controls.

(ii) preventing, reducing, curing, accelerating cure or healing, or reducing a bacterial infection, severity of infections or the ability of organisms to cause symptoms in a host or patient. (iii) relieving the signs or symptoms resulting from the disease or condition, e.g., reducing inflammatory lesions associated with bacterial infection.

In one specific embodiment there is provided a method of treating acne comprising administering to a subject in need thereof an effective amount of a composition that comprises an aminoglycoside-peptide conjugate (P-L-A) as described herein.

Propionibacterium acnes (P. acnes) are major etiological factors in acne vulgaris, a chronic human skin disorder. Although antibiotics are a common form of treatment against acne, the difficulties inherent to effective antimicrobial penetration in sebum and selective antimicrobial action in skin are recently compounded by increasing resistance of P. acnes clinical isolates.

In vitro studies have shown that although aminoglycosides are usually potent antimicrobials, P. acnes are not strongly susceptible to them, and aminoglycosides show limited effectiveness as oral antibiotics in vivo. As P. acnes are anaerobic bacteria, it is hypothesized that their intrinsic resistance is a result of poor aminoglycoside uptake, not a lack of ribosomal activity. The aminoglycoside- peptide conjugates describe herein equip aminoglycosides with cell-penetrating abilities. They are effective against slow-growing bacteria like P. acnes. Moreover, the AMP-like membrane activity will add an extra dimension of selectivity to the specific mechanisms inherent to aminoglycosides.

An aminoglycoside-peptide conjugate (e.g., Pentobra) of the present disclosure shows potent and selective bactericidal activity against P. acnes., but not against human skin cells in vitro. In direct comparison, Pentobra demonstrated bactericidal activity and drastically outperformed free tobramycin (by 5-7 logs) against multiple P. acnes strains. Moreover, electron microscopy (EM) studies showed that Pentobra had robust membrane activity, as treatment with Pentobra killed P. acnes cells and caused leakage of intracellular contents. In addition, Pentobra may also have potential anti-inflammatory effects as demonstrated by suppression of some P. acnes-induced chemokines. Importantly, the killing activity was maintained in sebaceous environments as Pentobra was bactericidal against clinical isolates in comedones extracts isolated from human donors. Thus, one specific embodiment relates to a method of treating acne vulgaris in a human subject comprising topically administering a therapeutically effective amount of a P-L-A conjugate, as defined herein.

Another specific embodiment relates to a method of decreasing inflammatory lesions of acne in a human subject comprising topically administering a therapeutically effective amount of a P-L-A conjugate, as defined herein, to the inflammatory lesions.

It will be appreciated that the practice of the several embodiments of the present invention will employ, unless indicated specifically to the contrary, conventional methods in virology, immunology, microbiology, molecular biology and recombinant DNA techniques that are within the skill of the art, and many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.(2009); Ausubel et al., Short Protocols in Molecular Biology, 3 rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used in this specification and the appended claims, the singular forms“a,” “an” and“the” include plural references unless the content clearly dictates otherwise. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise. Equivalents

While particular steps, elements, embodiments and applications of the present invention have been shown and described herein for purposes of illustration, it will be understood, of course, that the invention is not limited thereto since modifications may be made by persons skilled in the art, particularly in light of the foregoing teachings, without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. Further aspects and embodiments of the invention are disclosed in the following examples. EXAMPLES

Example 1: Comparison between Pentobra and tobramycin

A 12 AA peptide, called Pen, was designed and connected to tobramycin to produce a membrane-active antibiotic-peptide conjugate (MAAPC) hybrid, Pentobra. Small-angle x-ray scattering experiments on small unilamellar vesicles with lipid compositions mimicking bacterial cytoplasmic membranes showed that Pentobra can generate saddle-splay curvature in model bacterial membranes (Schmidt NW, et al. ACS Nano. 2014;8(9):8786-93), in a manner similar to antimicrobial peptides (AMPs). Conversely, tobramycin could not induce cubic phase formation in vesicles. These results were consistent with permeabilization experiments on bacteria. Single micromolar concentrations of Pentobra readily permeabilized the cytoplasmic membranes of E. coli, whereas permeabilization profiles from concentrations of tobramycin well above the minimum inhibitory concentration (MIC) (>10x) were indistinguishable from background. The generation of saddle-splay membrane curvature and permeabilization of bacteria membranes by Pentobra supports the approach of using saddle-splay curvature design rules to renovate existing antibiotics by giving them the additional function of membrane activity (PCT Application No. PCT/US2015/044897, filed 08/2015). The ability of Pentobra to permeate bacterial membranes suggests that MAAPCs may have therapeutic value in situations where aminoglycoside antibiotics are not effective.

To examine whether MAAPCs might have therapeutic value, plate killing assays comparing the activities of Pentobra and tobramycin against persistent bacteria were conducted. Remarkably, Pentobra showed dose-dependent bactericidal activity against both model Gram-negative (E. coli) and Gram-positive (S. aureus) persisters with reductions reaching 4-6 logs at the highest drug concentrations tested, while equivalent molar concentrations of tobramycin were not bactericidal (Schmidt NW, et al. ACS Nano 2014;8(9):8786-8793). Moreover, Pentobra is non-cytotoxic to eukaryotic cells (Schmidt NW, et al. ACS Nano 2014;8(9):8786-8793; Schmidt NW, et al. J Invest Dermatol 2015;135(6):1581-1589). These results show that equipping aminoglycosides with membrane activity is a promising approach to eradicate pathogenic organisms that are recalcitrant to existing therapies. Example 2: Antimicrobial activity of Pentobra against anaerobic pathogens

One shortcoming of aminoglycoside antibiotics is that, in vitro, they are not active against anaerobic bacteria. It is hypothesized that the lower PMF across the membranes of anaerobes impairs internalization (Davis BD, et al. Proceedings of the National Academy of Sciences 1986;83(16):6164-6168; Taber HW, et al. Microbiol Rev 1987;51(4):439), since aminoglycosides bind the ribosomes of anaerobic bacteria and ones that use oxygen with similar affinities (Bryan L, et al. Antimicrob Agents Chemother 1979;15(1):7-13). The issue is thought to be drug uptake. To determine if additional membrane activity could expand the activity spectrum of aminoglycosides to anaerobic bacteria, the activity of Pentobra against Propionibacterium acnes (Wang WLL, et al. Antimicrob Agents Chemother 1977;11(1):171-173; Dreno B, et al. European Journal of Dermatology 2004;14(6):391-399), a major etiological agent in acne (Ross J, et al. Brit J Dermatol 2003;148(3):467-478; McInturff JE, et al. J Invest Dermatol 2005;125(2):256-263) and the dominant pathogen in prosthetic shoulder implant infections (Achermann Y, et al. Clinical Microbiology Reviews 2014;27(3):419-440) was examined. Pentobra showed potent bactericidal activity (4- 6 log unit decrease in cfu) against P. acnes clinical isolates in standard liquid culture killing assays and in assays utilizing human comedone extracts that are more representative of the lipid-rich environment of human skin (Schmidt NW, et al. J Invest Dermatol 2015;135(6):1581-1589). Conversely, both Pen peptide and tobramycin alone were much less effective. These results suggest that MAAPCs might have therapeutic value against anaerobic pathogens and they have potential as topical antimicrobial agents. Example 3: Synthetic approach of MAAPCs

MAAPCs 02-06 were synthesized based on a similar method used for the prototype Pentobra [1, 2], but with different peptide sequences and/or different aminoglycosides (tobramycin, neomycin and kanamycin). In addition, an analogue of Pentobra named cPentobra (MAAPC07) was synthesized using a different synthetic approach. While the same peptide sequence and same aminoglycoside (tobramycin) were used as for Pentobra, tobramycin was conjugated to the C-terminal of the peptide through a 1,2,3-triazole ring using“click” chemistry (Figure 3). Our approach is not limited to aminoglycosides as this approach may be used to renovate other classes of antibiotics such as β-lactams (penams, cephems, monobactams, carbapenems), macrolides, quinolones, tetracyclines, phenicols and sulfonamides. Figure 1 shows some examples of new conjugates composed of a membrane active peptide (pre-defined according to our design rules, see PCT Application No. PCT/US2015/044897, filed 08/2015) attached to ampicillin (or other aminopenicillins such as amoxicillin) using a thiol-maleimide coupling. For those examples, an extra cysteine (bearing a thiol on its side chain) is incorporated either at the N-terminal or C-terminal or between two amino acids of the peptide sequence; concurrently, ampicillin is modified with a maleimide group on its primary amine to allow a thiol– maleimide coupling with the cysteine-modified peptide. This list is not exhaustive and can be extended to other antibiotics using different spacers, linkers and synthetic approaches. The coupling reactions include known methods in the art, such as amide bond formation (e.g., via succinimide coupling), reductive amination, “click” chemistry, thiol-maleimide coupling at either the N-terminal or C-terminal ends of the peptide after modification with appropriate functionality of both, the peptide and the antibiotic. Coupling can take place either in solution or solid phase. Example 4: Bacteriology data

The antimicrobial activity of various example MAAPCs is provided, including minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays (Table 2-4), hemolytic activity against human red bloods (hRBCs) (Table 5), killing assays, and biofilm inhibition assays (Figure 2). cPentobra exhibited bactericidal properties against stationary and persistent Pseudomonas aeruginosa (Figure 2A and B) and was capable of inhibiting P. aeruginosa biofilm growth (Figure 2C). cPentobra outperformed tobramycin and had better activity than Pentobra. It is worth noting that the antimicrobial activity observed when the Pen peptide is mixed with tobramycin (but not chemically conjugated) was similar to tobramycin alone against P. aeruginosa persisters indicating that the synergistic effect occurs only when the antimicrobial peptide and tobramycin are chemically attached as in Pentobra or cPentobra.

While Pentobra (MAAPC01) and MAAPC04 induced significant permeabilization of the outer membrane (OM) of E. coli D31 at a concentration lower than MICs (2 μM), the other MAAPCs displayed relatively low OM permeabilization, and as expected tobramycin had no effect (Figure 4A). With the exception of MAAPC06, all MAAPCs disrupted, with different degrees of susceptibility, the inner membrane (IM) of E. coli ML35 at a concentration of 2 μM (Figure 4B). Pentobra (MAAPC01) and cPentobra (MAAPC07) exerted the highest permeation effect, which was comparable to the positive control (melittin). Example 5: Materials and methods

Synthesis of MAAPC02-MAAPC06

The MAAPCs (02-06) (Table 1) were synthesized on a similar method as previously described for Pentobra [1]. Briefly, the peptide sequence with protected side chains is synthesized by solid phase synthesis on 2-chlorotrityl chloride resin either manually or automatically using an automated peptide synthesizer. Concurrently, the amine groups of the aminoglycoside (tobramycin, kanamycin or neomycin) are protected with t-butyloxycarbonyl (Boc) groups to provide Boc- protected aminoglycoside. Next, the primary hydroxyl of Boc-protected aminoglycoside is selectively reacted with succinic anhydride to introduce a terminal carboxyl function allowing the coupling with the N-terminal group of the fully protected and resin-anchored peptide. Finally, the conjugate is cleaved off the resin, fully deprotected by treatment with trifluoroacetic acid mixture containing scavengers and purified by reversed-phase high performance liquid chromatography (HPLC). Synthesis of MAAPC07 (cPentobra)

Synthesis of tosyl-Boc 5 -tobramycin

Boc 5 -tobramycin synthesized as previously described [1] (3.58 g, 3.70 mmol, 1 eq.) was dissolved in pyridine (85 mL) and cooled down with an ice bath. p- toluenesulfonyl chloride (987.5 mg, 5.18 mmol, 1.5 eq.) was added to the mixture. After stirring for 2 h at room temperature, p-toluenesulfonyl chloride (987.5 mg, 5.18 mmol, 1.5 eq.) was added again. The solution was stirred for 12 h at room temperature under argon. The solution was concentrated via rotary evaporation, diluted with ethyl acetate (200 mL), washed with non-saturated brine (3 × eq. vol.), dried with MgSO 4 , and concentrated to dryness via rotary evaporation. The white solid was purified by silica gel chromatography to yield 1.48 g (36%). Mass analysis (MALDI-TOF): m/z 1144.5 (calcd for C50H83NaN5O21S [M+Na] + m/z 1144.519). Synthesis of Boc 5 -tobramycin-azido

Tosyl-Boc5-tobramycin (1.48 g, 1.32 mmol, 1 eq.) was dissolved in DMF (19 mL). Sodium azide (858 mg, 13.2 mmol, 10 eq.) was added to the mixture. The solution was stirred at 80°C for 12 h under argon. The solution was diluted with ethyl acetate (200 mL), washed with DI water (3 × eq. vol.), dried with MgSO4, and concentrated to dryness via rotary evaporation to yield 1.30 g (99%) of a white solid. Mass analysis (MALDI-TOF): m/z 1015.5 (calcd for C 43 H 76 NaN 8 O 18 [M+Na] + m/z 1015.517). Synthesis of Fmoc-Pra(BocTobra)-OH by Click Chemistry

Fmoc-Pra-OH (188.2 mg, 0.56 mmol, 1 eq., AAPPTEC) and Boc5-tobramyci- azido (612.7 mg, 0.62 mmol, 1.1 eq.) were dissolved in DMF (20 ml) at 40°C under argon. 1 mL of copper sulfate aqueous solution (0.28 mmol, 0.5 eq.) and 1 ml of sodium ascorbate aqueous solution (0.56 mmol, 1 eq.) were added to the mixture. The solution was stirred at 40°C for 24 h. The solution was concentrated via rotary evaporation, diluted with ethyl acetate (200 mL), washed with water (3 × eq. vol.), dried with MgSO 4 , and concentrated to dryness via rotary evaporation. The white solid was purified by silica gel chromatography to yield 453 mg (61%). Mass analysis (MALDI-TOF): m/z 1350.591 (calcd for C 63 H 93 NaN 9 O 22 [M+Na] + m/z 1350.633). Synthesis of cPentobra The (RQIKIWFQNRRWPra(Tobra)) peptide (cPentobra) was synthesized by Fmoc/t-butyl batch solid phase synthesis on an Applied Biosystems 433A automated peptide synthesizer, which allowed for direct conductivity monitoring of Fmoc deprotection. A 0.2 mmol scale synthesis was conducted using a pre-loaded 2- chlorotrityl resin. Loading of the first amino acid. A solution of Fmoc-Pra(BocTobra)-OH (448 mg, 0.34 mmol, 1 eq.) and DIEA (235 mL, 7.0 mmol, 4 eq.) in dry DCM (10 mL) was added to 2-chlorotrityl chloride resin (340 mg, 0.34 mmol, 1 eq.) and the reaction stirred for 4 h. The resin was transferred into a peptide vessel fitted with a polyethylene filter disk, and washed with a solution of DCM/MeOH/DIEA (17:2:1; 3 × 20 mL), DCM (3 × 20 mL), DMF (2 × 20 mL), and DCM (2 × 20 mL). The grafting yield was determined by measuring the absorbance of N-(9- fluorenylmethyl)piperidine complex at 301 nm by UV-vis spectroscopy (after treatment with piperidine) and resulted 0.35 mmol/g. Automatic synthesis of peptide. Subsequent Fmoc amino acids were coupled using a“conditional double coupling” protocol on a 0.2 mmol scale. The Fmoc group was cleaved from the peptide-resin using a piperidine solution and monitored by conductivity. Subsequent amino acids (5 eq. amino acid) were activated with a mixture of HBTU/HOBt/DIEA and attached to the N-terminal of the peptide-resin. Cleavage of the peptide from the resin with removal of the acid-labile protecting groups. This was achieved by using 10 mL of a scavenging mixture of TFA/phenol/water/thioanisole/TIS (10/0.75/0.5/0.5/0.25 v/w/v/v/v) for 3 h. The resin was filtered out with a fritted filter, rinsed with 1 mL of TFA and 20 mL of DCM, the filtrate containing the unprotected peptide was concentrated to small volume and the product was precipitated with cold diethyl ether, isolated by filtration and dried under vacuum overnight. The peptide was purified by preparative RP-HPLC (JASCO system) at 17 mL/min on a Waters C18 column (250 × 22 mm, 5 mm) using a gradient of A [H2O + 0.1% TFA] and B [CH3CN + 0.1% TFA]: 0% of B for 5 min, 0%→70% for 7 min, 70% for 3 min, 70%→100% for 2 min and 100% for 2 min; detection at 214 nm; t r = 10.4 min. CH 3 CN was evaporated under reduced pressure and the aqueous solution was freeze-dried to give a white solid (211.5 mg, 46%). Mass analysis (MALDI-TOF): m/z 2318.519 (calcd for C104H165N36O25 [M+H] + m/z 2318.275). ABBREVIATIONS

Abbreviations: Boc: tert-butyloxycarbonyl; CH 3 CN: acetonitrile; DCM: dichloromethane; DIEA: N,N-diisopropyl-N-ethylamine; DMF: dimethylformamide; Fmoc: 9-fluorenylmethoxycarbonyl; HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate; HOBt: N-hydroxybenzotriazol; MALDI- TOF: matrix assisted laser desorption ionization time of flight; MeOH: methanol; NMP: N-methyl-2-pyrrolidone; RP-HPLC: reversed-phase high performance liquid chromatography; TFA: trifluoroacetic acid; TIS: triisopropylsilane. MICROBIAL MEMBRANE DISRUPTING PEPTIDE SEQUENCES GWIRNQFRKIWQR (SEQ ID NO: 1)

GWRRNQFWIKIQR (SEQ ID NO: 2)

GPWWFKWPRLI (SEQ ID NO: 3)

GFHGVKNLARRIL (SEQ ID NO: 4)

GWRNQIRKGWQR (SEQ ID NO: 5)

CFHRLFKRILRK (SEQ ID NO: 6)

RWWRLI (SEQ ID NO: 7)

RWRWIR (SEQ ID NO: 8)

RQIKIWFQNRRW (SEQ ID NO: 9)

RQIKIWFQNRRWPra (SEQ ID NO: 10) *Pra = Propargylglycine (glycine with an alkyne function for click conjugation) TABLES

Table 2. MICs and MBCs of MAAPCs against E. coli. MICs and MBCs of the peptide, the antibiotic and a mixture of the peptide and antibiotic are also included.

Table 3. MICs and MBCs of MAAPCs against P. aeruginosa. MICs and MBCs of the peptide, the antibiotic and a mixture of the peptide and antibiotic are also included.

Table 4. MICs and MBCs of MAAPCs against S. aureus. MICs and MBCs of the peptide, the antibiotic and a mixture of the peptide and antibiotic are also included.

Table 5. Hemolytic activity of MAAPCs on human red blood cells (hRBCs) and selectivity of MAAPCs against bacteria over hRBCs.

Table 6. MIC of MAAPCs against different bacteria.

REFERENCES

Note: This application references a number of different publications as indicated throughout the specification by reference numbers enclosed in brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further, the actual publication dates may be different from those shown and require independent verification. [1] Schmidt NW, Deshayes S, Hawker S, Blacker A, Kasko AM, Wong GC. Engineering persister-specific antibiotics with synergistic antimicrobial functions. ACS Nano.2014;8(9):8786-93.

[2] Schmidt NW, Agak GW, Deshayes S, Yu Y, Blacker A, Champer J, Xian W, Kasko AM, Kim J, Wong GCL. Pentobra: A Potent Antibiotic with Multiple Layers of Selective Antimicrobial Mechanisms against Propionibacterium Acnes. J Invest Dermatol.2015;135(6):1581-9.

CONCLUSION

This concludes the description of the preferred embodiment of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.