WO2020023620A1 - Procédés et compositions de bibliothèques de bactériophages modifiés chimiquement - Google Patents

Procédés et compositions de bibliothèques de bactériophages modifiés chimiquement Download PDF

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WO2020023620A1
WO2020023620A1 PCT/US2019/043211 US2019043211W WO2020023620A1 WO 2020023620 A1 WO2020023620 A1 WO 2020023620A1 US 2019043211 W US2019043211 W US 2019043211W WO 2020023620 A1 WO2020023620 A1 WO 2020023620A1
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apba
peptide
phage
phage display
residue
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PCT/US2019/043211
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English (en)
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Jianmin Gao
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Trustees Of Boston College
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof

Definitions

  • the present invention is directed to novel chemically modified phage libraries.
  • Antibiotic resistant bacterial pathogens have become a global threat to public health. Diverse mechanisms of resistance of essentially all current antibiotics have been elucidated in recent years and are continuously being discovered. According to the Center for Disease Control, over 2 million antibiotic-resistant infections are reported each year leading to approximately 23,000 deaths in the United States alone. Although resistance occurs naturally, misuse and overuse of broad-spectrum antibiotics in humans and animals accelerates the process. There is thus an urgent need for a change in the way antibiotics are utilized along with the development of novel antimicrobial agents. Ideally, to avoid unintentional elicitation of antibiotic resistance, it would be advantageous to replace widely used broad-spectrum antibiotics with narrow-spectrum antibiotics. Furthermore, the use of broad-spectrum antibiotics can cause undesirable disruptions to the microbiota, which plays critical roles in various aspects of human biology.
  • phage-display technology was limited to presenting peptides only composed of natural, proteinogenic amino acids. Due to technological advances in the field, phage libraries can now be chemically and genetically modified to present unnatural entities, which greatly expand the chemical space of phage displayed molecules. For example, selective cysteine alkylation has been utilized to create bicyclic peptide libraries on phage. Similarly, a glycopeptide library has been developed through oxidative cleavage of an N- terminal serine or threonine that yields a bioorthogonal aldehyde handle for conjugation to carbohydrates.
  • Antibiotic resistance of bacterial pathogens poses an increasing threat to the wellbeing of society and urgently calls for new strategies for infection diagnosis and antibiotic discovery.
  • the antibiotic resistance problem to a large extent arises from extensive use of broad-spectrum antibiotics.
  • a narrow-spectrum antibiotic that specifically targets and kills the disease causing strain. This is particularly important considering the commensal bacterial species that are beneficial and sometimes even critical to the health of a human being.
  • the present disclosure provides a novel phage display library that incorporates chemical modification of phage displayed peptides.
  • the chemical modification motifs are dynamic covalent binding motifs.
  • the covalent binding motif is a pair of 2-acetylphenylboronic acid (APB A) moieties that are installed onto phage displayed peptides to bind biological amines via dynamic formation of iminoboronates.
  • APB A 2-acetylphenylboronic acid
  • the present disclosure provides chemical modification of phage displayed peptides yields an APBA dimer library.
  • the display peptides of the APBA dimer phage library are linear peptides.
  • the display peptides of the APBA dimer phage library are cyclic and/or multicyclic peptides wherein crosslinks are introduced to the linear peptide architecture to general cyclic and/or multicyclic peptides. These cyclic and/or multicyclic peptide libraries maximize the chance of success for a diverse range of bacterial pathogens.
  • the present disclosure provides that the APBA dimer phage library can be extended to discover binders of various bacterial pathogens.
  • screening of the iminoboronate-capable APBA dimer library of the present disclosure against live bacterial cells yielded potent and selective binders of Staphylococcus aureus as well as a colistin- resistant strain of Acinetobacter baumannii.
  • the present disclosure further provides that these bacterial binders identified from the APBA dimer phage library can be readily converted to targeted antibiotics that specifically eradicate the corresponding strain of bacteria.
  • the iminoboronate-capable APBA dimer phage library provided herein serves as a powerful tool to advance the development of narrow-spectrum antibiotics.
  • the present disclosure further provides that antibiotics of other modes of action, such as those targeting cell membranes, including but not limited to, vancomycin and daptomycin, can be utilized to build peptide-antibiotic conjugates for effective and targeted bacterial cell killing.
  • antibiotics of other modes of action such as those targeting cell membranes, including but not limited to, vancomycin and daptomycin
  • the chemically modified phage library described herein provides a convenient way to screen live intact bacterial for potential target, and can also be readily adapted to other targets including mammalian cells.
  • the present disclosure provides the iminoboronate-capable APBA dimer phage libraries that provide advancement in bacterial imaging reagents or novel antibiotics.
  • the disclosed APBA dimer phage display libraries are applicable for drug screening for selection of functional molecules towards a variety of different targets.
  • the disclosed APBA dimer phage display libraries can also be used for the development of novel protein inhibitors.
  • the present disclosure provides a new paradigm for the development of targeted antibiotics and/or peptide-antibiotic conjugates.
  • Figures 1A-1D show modification of Ph.D.-C7C library with APBA warheads.
  • A illustration of a modified phage binding to bacterial cells via iminoboronate formation.
  • B Illustration of the cysteine labeling strategy to display 2-APBA on phage (SEQ ID NOS: 268-269, respectively, in order of appearance).
  • C Structure of the chemical modifiers of the C7C phage library.
  • D Confirmation of APBA labeling of phage via fluorescent gel imaging after conjugation with Scz-FITC.
  • Lane 1 ladder imaged at 660nm; Lane 2: ladder imaged at 495nm; Lane 3 : reduced phage; Lane 4: Biotin-IA labeled phage; and Lane 5: 2-APBA labeled phage.
  • FIGS 2A-2D show phage display against S. aureus.
  • A Schematic representation of panning against live bacterial cells (SEQ ID NOS: 271-274, respectively, in order of appearance). Stars denote APBA-IA modification.
  • B Flow cytometry analysis of fluorescein labeled KAM5 in for staining S. aureus cells. Data of replicate experiments are given in Figures 14A-14C, which shows consistent results.
  • C Flow cytometry comparison of KAM5 to KAM5 Cyclic (no APBA modification) and a naive APBA dimer peptide KAM6.
  • Figures 3A-3B show selective binding of KAM5 to S. aureus over other bacterial species.
  • B. subtilis & E. coli were used as representative gram-positive and gram-negative bacteria species.
  • A Results of flow cytometry analysis with fluorescein labeled KAM5 in the presence of 1 mg/mL BSA. Replicate data set is given in Figure 14, which shows consistent results.
  • B Results of microscopy analysis using TAMRA labeled KAM5 (10 pM). Scale bar: 10 pm.
  • Figures 4A-4D show conjugation of a phototoxin to induce targeted killing of S. aureus.
  • A Cartoon representation of photodynamic therapy with KAM5-Eosin.
  • B Structure of KAM5- Eosin (SEQ ID NO: 277).
  • C Percent killing of S. aureus by KAM5-Eosin and controls with and without photoirradiation.
  • D Percent killing of several bacterial species with KAM5-Eosin (2 pM) to highlight the S. aureus specificity.
  • FIGS 5A-5D show phage display to discover peptide probes for a LOS- strain of A. baumannii (AB5075).
  • A Flow cytometry analysis of the representative peptide hit KAM8 in staining A. baumannii (LOS- vs LOS+) in the presence of 1 mg/mL BSA. Replicate data set is presented in Figures 14A-14C, which shows consistent result.
  • B A. baumannii cell staining examined by microscopy with TAMRA labeled KAM8 at 2 mM for LOS- and 10 pM for LOS+. Scale bar: 10 pm.
  • C Percent cell killing of A. baumannii (LOS-) with and without photoirradiation.
  • D Percent cell killing of the LOS+ versus LOS- strains of A. baumannii with 2 pM KAM8-Eosin. The contrasting outcomes of these strains highlight the high strain specificity of KAM8.
  • Figure 6 shows the synthetic scheme of APBA-IA (5).
  • Figure 7 shows the 1 H-NMR of APBA-IA (5).
  • Figure 8 shows 13 C-NMR of APBA-IA (5).
  • Figure 9 shows pulse-chase confirmation of APBA-IA labeling on library phage.
  • Figure 10 shows fluorescence microscopy studies of phage binding to S. aureus assessed using anti-Ml3 antibody (FITC labeled). Each phage variant is designated as the first three letters of the heptapeptide sequence. Scale bar: 10 pm.
  • Figures 11A-11B show (A) structure and (B) LC-MS characterization of KAM5 with a fluorescein label. The results are shown as an example to demonstrate the purity and integrity of the peptides used for this study.
  • Figures 12A-12D show flow cytometry analysis of S. aureus staining by KAM1 (A), KAM2(B), KAM3(C), and KAM4 (D) in presence and absence of BSA.
  • Figures 13A-13B show flow cytometry analysis of S. aureus staining by KAM3 (A) and KAM5 (B) up to 10 pM in concentration. Results are shown for with and without BSA.
  • Figures 14A-14C show duplicate flow cytometry experiments demonstrating consistent results between trials. Data sets on the left are presented in the main text figures while their duplicate datasets are presented on the right.
  • A S. aureus staining by KAM5 (Fig. 2B and Duplicate)
  • B Bacterial species selectivity studies of KAM5 (Fig. 3A and Duplicate)
  • C A. baumannii staining by KAM8 (Fig. 5A and Duplicate).
  • Figure 15 shows concentration profile of KAM5 staining S. aureus in comparison to that of a previously reported peptide for S. aureus labeling (Hlys-ABl). All samples were prepared to have 1 mg/mL BSA.
  • Figures 16A-16B show further evaluation of the protein-enhanced bacterial staining by KAM5.
  • A Flow cytometry comparison of HSA and BSA in enhancing KAM5 binding to S. aureus.
  • B Assessing protein binding of KAM5 using fluorescence anisotropy, for which various concentrations of BSA or HSA were delivered to a 96-well plate (Corning 3603) in PBS (pH 7.4).
  • Figure 17 shows microscopic images of S. aureus and MRS A treated with TAMRA labeled KAM5 at 2 mM concentration. Scale bar: 10 pm.
  • FIG. 18 shows flow cytometry analysis of KAM14-17 for S. aureus staining in comparison to KAM5. The results clearly show the superior potency of the iminoboronate-capable peptide KAM5 for labeling S. aureus cells. *Note: KAM14 was only analyzed up to 3 pM due to aggregation at higher concentrations.
  • Figure 19 shows KAM5-Eosin showing comparable potency for killing MRS A versus the strain of S. aureus used in phage selection of KAM5.
  • Figures 20A-20B show MTT assay to assess mammalian cell toxicity on Jurkat (A) and HEK293T (B) cells after treatment for 24 hrs. Note that, for HEK 293T cells, photoirradiation alone resulted in some extent of cell killing. However, the peptide addition elicited no additional cell killing indicating lack of toxicity.
  • Figures 21A-21B show A. baumannii (LOS-) staining by KAM7-10.
  • A Flow cytometry analysis of KAM7-10 staining of A. baumannii (LOS-) in presence and absence of BSA. Fluorescein labeled peptides were used for this analysis.
  • B Comparison of A. baumannii (LOS-) staining by KAM8 to KAM8-Cyclic (precursor of KAM8, no APBA conjugated) and a naive APB A dimer KAM6 at 1 pM in the presence of 1 mg/mL BSA.
  • Figure 22 shows fluorescence microscopy studies of KAM8 (2 pM) staining several bacterial species. TAMRA labeled peptide was used for this study. All samples contain 1 mg/mL BSA. Scale bar: 10 pm.
  • Figures 23 A-23B show (A) phage displayed cyclic peptides carrying 2-APB A warhead and (B) a proposed route for synthesis of APBA-blA.
  • Figures 24A-24B show iminoboronate-mediated peptide cyclization and bicyclization.
  • A APBA-IA that can be used for modification of a Cys side-chain.
  • B Example of peptide bicyclization with between each of two Cys residues modified with APBA-IA and each of two Lys pairs strategically placed in the peptide sequence (SEQ ID NO: 282). For each of the Cys and Lys residues in the peptide sequence shown, the side chains for each are discretely shown to provide detailed information on the interactions and reactions involved in the bicyclization reaction.
  • Figure 25 shows titering results of M13 phage treated with NaCNBFF of varied concentration and time. The results indicate that the NaCNBFE treatment minimally compromises the phage’s viability.
  • Figure 26 shows peptide bicyclization and further functionalization on phage (SEQ ID NOS 278-281, respectively, in order of appearance).
  • Figure 27 shows structures of bactericidal agents for conjugation to bacteria-specific peptide probes. Highlighted in red are primary amines that can serve as conjugation site.
  • Figure 28 shows the general concept of phage libraries that display dynamic covalent binding motifs. Such dynamic covalent binding motifs can interact with a biological target through unprecedented mechanisms.
  • 2-APBA is one example of a dynamic covalent binding motif, as shown in these figures.
  • the molecules engage with a biological target via conjugation to amines (e.g., lysine side chains).
  • Dynamic covalent binding motifs may also bind other functionalities such as cysteine and serine side chains.
  • Figure 30 shows the fundamental properties of dynamic covalent binding. Specifically, it shows the advantage of 2-APBA (in comparison to the control molecule) for binding biological amines with enhanced potency.
  • Figure 31 shows the lipid modifications (with amines) that give rise to antibiotic resistance.
  • the present disclosure provides novel chemically modified phage libraries and applications and/or use of such chemically modified phage libraries.
  • the present disclosure provides the construction and validation of a phage library displaying reversible covalent binding motifs incorporated onto the display peptides.
  • chemical modification of phage displayed peptides yields an APBA dimer library, which allows facile screening against bacterial cells to identify reversible covalent binders of specific bacterial strains.
  • the display peptides are linear peptides.
  • the display peptides are cyclic and/or multicyclic peptides in which crosslinks are introduced to the linear peptide architecture to generate cyclic and/or multicyclic peptides.
  • the APBA dimer phage libraries with the cyclic and/or multicyclic peptides maximize the chance of success for a diverse range of bacterial pathogens.
  • the present disclosure further provides use of the novel chemically modified phage libraries for drug screening for functional molecules toward a variety of different targets.
  • the present disclosure provides a drug screening method of screening the chemically modified phage libraries with live bacterial cells which leads to develop bacterial imaging reagents, as well as novel antibiotics.
  • the present disclosure provides the use of the chemically modified phage libraries for quick discovery of targeted antibiotics, which, in comparison to the currently used broad-spectrum antibiotics, could reduce the unnecessary cultivation of antibiotic resistance and minimize the disruption of host microbiota.
  • the present disclosure provides novel chemically modified phage libraries for advancement in bacterial targeting and/or imaging reagents, and or novel antibiotics, as well as for novel protein inhibitors and/or for peptide-antibiotic conjugates.
  • the present disclosure thus provides a new paradigm for such development of targeted antibiotics and/or peptide-antibiotic conjugates for effective and targeted bacterial cell killing.
  • ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. For example, if the value“10” is disclosed, then“about 10” is also disclosed. Ranges can be expressed herein as from“about” one particular value, and/or to“about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms a further aspect. For example, if the value“about 10” is disclosed, then“10” is also disclosed.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure.
  • the upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range.
  • the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
  • the stated range includes one or both of the limits
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase“x to y” includes the range from‘x’ to‘y’ as well as the range greater than‘x’ and less than‘y’.
  • the range can also be expressed as an upper limit, e.g.‘about x, y, z, or less’ and should be interpreted to include the specific ranges of‘about x’,‘about y’, and ‘about z’ as well as the ranges of‘less than x’, less than y’, and‘less than z’.
  • phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of‘about x’,‘about y’, and‘about z’ as well as the ranges of‘greater than x’, greater than y’, and‘greater than z’.
  • phrase“about‘x’ to‘y’”, where‘x’ and‘y’ are numerical values, includes“about‘x’ to about‘y’”.
  • a numerical range of“about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • an amount, size, formulation, parameter or other quantity or characteristic is“about,”“approximate,” or“at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or“at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
  • the terms“optional” or“optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • the terms“reversible covalent binding motif,”“reversible covalent binding warhead,” and“reversible covalent binding motif and/or warheads” can be used interchangeably and refer to a peptide comprising a two cysteine moieties, wherein each cysteine moiety is covalently linked to an APBA moiety such that the peptide can bind to a target molecule or target cell, e.g., an microbe or bacteria comprising a target molecule, through a combination of non- covalent interactions involving the peptide backbone and amino acid side-chains and reversible covalent interactions comprising a reversible covalent linkage between one or both of the APBA moieties and a moiety, e.g., an amine group, in the target molecule or target cell.
  • a peptide comprising a reversible covalent binding motif can be within a peptide that is in a phage display library or an isolated peptide, e
  • alkyl as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t- butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • the alkyl group can be cyclic or acyclic.
  • the alkyl group can be branched or unbranched.
  • the alkyl group can also be substituted or unsubstituted.
  • the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.
  • A“lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms.
  • alkyl group can also be a Cl alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, Cl- C10 alkyl, and the like up to and including a C1-C24 alkyl.
  • APBA APBA residue
  • APBA moiety a chemical residue comprising an APBA structure given by the following formula:
  • APB A residue is a structure given by the following formula:
  • APBA-IA refers to a compound comprising an APBA residue and an iodoacetamide residue having a structure given by the following formula:
  • a 1 and A 2 are independently a C1-C6 alkyl.
  • a particular example of APBA-IA is (2-acetyl-5-(3-(2-iodoacetamido)propoxy)phenyl)boronic acid, that is, a compound having a structure given by the following formula:
  • amino acid as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids.
  • Standard amino acid means any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid residue means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source.
  • synthetic amino acid also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions.
  • Amino acids contained within the peptides of the present disclosure, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half-life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention.
  • APBA modifiable dimer phage library refers to a phage display library comprising peptides expressed on the surface of the phage display library comprising two cysteine residues that can be chemically modified with an APBA moiety.
  • APBA dimer phage library refers to a phage display library comprising peptides expressed on the surface of the phage display library comprising two APBA modified cysteine residues.
  • a“therapeutic APBA peptide” is a peptide comprising a peptide sequence as disclosed herein such that the peptide comprises two APBA modified cysteine residues.
  • APBA modified cysteine residue refers to a cysteine residue comprising an APBA moiety. That is, an APBA modified cysteine residue has a structure given by the formula:
  • a modified cysteine residue can is a structure given by the formula:
  • APBA modified peptide is a peptide comprising an APBA modified cysteine residue.
  • amino acid is used interchangeably with“amino acid residue,” and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
  • Amino acids have the following general structure:
  • R is a“side chain” or“side group” of the amino acid.
  • Amino acids may be classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • peptides or peptide compounds of the present disclosure follows the conventional practice wherein the amino residue is presented to the left and the carboxy group to the right of each amino acid residue.
  • a peptide sequence comprising a sequence of amino acid residues from the amino terminus to the carboxy terminus an alanine residue, an aspartic acid residue, a cysteine residue, and a glycine residue can be specified using the one-letter amino acid code as follows:
  • a peptide sequence the amino- and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified.
  • Subscripts in a peptide sequence can be used to indicate a repetition of amino acid, and a subscript range can be used to indicate that the indicated amino acid can be repeated for any of the number of instances of an integer specified by the range, inclusive of the upper and lower limits.
  • a peptide sequence can also be specified with variable positions using Xxx (three-letter code) or X (one-letter).
  • a peptide sequence comprising a sequence of amino acid residues from the amino terminus to the carboxy terminus an alanine residue, an aspartic acid residue, one to five amino acids selected from the standard amino acids, a cysteine residue, and a glycine residue can be specified using the one-letter amino acid code as follows:
  • an“analog” of a chemical compound is a compound that, by way of example, resembles another in structure but is not necessarily an isomer (e.g., 5-fluorouracil is an analog of thymine).
  • an amino acid can be represented by the full name thereof, by the three- letter code corresponding thereto, or by the one-letter code corresponding thereto.
  • the full name, three-letter code, and one-letter code for 20 standard amino acids are as indicated in the table below.
  • “basic” or“positively charged” amino acid refers to amino acids in which the R groups have a net positive charge at pH 7.0, and include, but are not limited to, the standard amino acids lysine, arginine, and histidine.
  • A“compound,” as used herein, refers to a polypeptide, an isolated nucleic acid, or other agent used in the method of the present disclosure.
  • the terms“polypeptide”,“peptide”, or“protein” refer to a series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • the amino acid residues are preferably in the natural“L” isomeric form.
  • residues in the“D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • the amino acids in addition to the 20 standard amino acids, include modified and unusual amino acids.
  • a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group.
  • APBA modified peptide refers to a peptide comprising two cysteine residue comprising an APBA moiety having the structure and sequences as disclosed herein.
  • target cell refers to a cell or cell-type that is to be specifically bound by a member of a phage display library of the present disclosure.
  • Target cells can be antibiotic resistant bacterial pathogens, e.g., Staphylococcus aureus or colistin-resistant strains of Acinetobacter baumannii , for which a binding peptide is sought.
  • the target cell is typically characterized by the expression a target molecule that is characteristic of the cell type, i.e., characteristic of the target cell in that it is uniquely expressed on the target cell compared to a non target cell or a target molecule that is overexpressed on the target cell compared to a non-target cell.
  • a target cell can be a cell, such as a Staphylococcus aureus or colistin- resistant strains of Acinetobacter baumannii , which expresses a target molecule, such as a protein or carbohydrate that can be bound by a phage display library.
  • a target molecule may be unique to the target cell compared to other cells or may be a molecule which is overexpressed by the target cell compared to other cells.
  • phage display refers to a method of using phage to heterologously express coat proteins or peptides for testing, e.g., particularly newly generated peptides (e.g., from about 5 to about 10 amino acids in length) thereof.
  • a gene encoding a protein or peptide is cloned and inserted into a phage genome or genetic material in such a way that the protein or peptide is displayed (i.e., expressed) on the surf ace of the phage, which is a recombinant phage.
  • Phage expressing peptides that interact with a target molecule or cell can be selected by selecting the protein or peptide directly using panning or affinity chromatography.
  • the non-bound phages can be removed by washing the cells expressing the target molecule.
  • the bound proteins or peptides produced by phages can then be isolated from the target molecule or target cell to which they are bound, and since they are still part of the phage, they can be grown in enough quantity to identify the gene sequence, and hence the protein sequence. This allows further manipulation of phages that bind to the target molecule(s).
  • phage display library refers to a collection of phage (e.g., filamentous phage) comprising collection of random sequences of nucleic acids that have been inserted into a phage vector, wherein the phage express a heterologous peptide encoded by the random sequences of nucleic acids therein on the surface of a phage particle.
  • the library can contain a few or a large number of random combinations of nucleic acid sequences, varying from about ten to several billion combinations of nucleotide sequences or more that code for a vast number ( ⁇ l0 12 ) of random peptides.
  • the external peptide is free to interact with (bind to) other moieties with which the phage are contacted.
  • Each phage displaying an external protein is a “member” of the phage display library.
  • a molecule or a phage vector can be linked to a tag, which can facilitate recovery or identification of the molecule.
  • the heterologously expressed proteins or peptides that are on the surface of a phage particle can be chemically modified to expand the chemical space encompassed by the phage display library, e.g., such a chemically modified phage display library is the disclosed APBA dimer phage library.
  • phage refers to a bacteriophage or virus that infects bacteria and is capable of displaying a heterologous polypeptide or peptide on its surface.
  • a phage comprises a protein coat or capsid enclosing the phage genome or genetic material (DNA or RNA) which is injected into a bacteria upon infection of the bacteria by the phage.
  • the injected genetic material directs the bacteria to synthesize the phage’s genetic material and proteins encoded by the phage genetic material using the host bacteria’s transcriptional and translational apparatus.
  • These phage components then self-assemble to form new phage viruses or particles.
  • the phage vector is, or is derived from, a filamentous bacteriophage, such as, for example, fl, fd, Pfl, Ml 3, etc.
  • the phage may contain a selectable marker such as tetracycline (e.g.,“fd-tet”).
  • tetracycline e.g.,“fd-tet”.
  • Various filamentous phage display systems are well known to those of skill in the art (see, e.g., Zacher et al. (1980) Gene 9: 127-140, Smith et al.(l985) Science 228: 1315-1317 (1985); and Parmley and Smith (1988) Gene 73 : 305-318).
  • phage vector is a bacterial virus which can receive the insertion of a gene or other genetic material, resulting in a recombinant DNA molecule.
  • the phage vector is capable of self-replication in a host organism.
  • a phage vector contains an origin of replication for a bacteriophage but not for a plasmid.
  • viral packaging signal refers a nucleic acid sequence necessary and sufficient to direct incorporation of a nucleic acid into a viral capsid.
  • assembly cell refers to a cell in which a nucleic acid can be packaged into a viral coat protein (capsid). Assembly cells may be infected with one or more different virus particles (e.g. a normal or debilitated phage and a helper phage) that individually or in combination direct packaging of a nucleic acid into a viral capsid.
  • virus particles e.g. a normal or debilitated phage and a helper phage
  • the term“detectable label” refers to any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g.
  • DynabeadsTM DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3H, 1251, 35S, 14C, or 32P
  • enzymes e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA
  • calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • Those detectable labels that can be expressed by nucleic acids are referred to as “reporter genes” or“reporter gene products”.
  • fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like.
  • CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281 : 2013-2016).
  • highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281 : 2016-2018).
  • a residue of a chemical species as herein refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species.
  • an example of a residue can be an ethylene glycol residue in a polyester refers to one or more -OCH2CH2O- units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester.
  • a sebacic acid residue in a polyester refers to one or more - CO(CH2)8CO- moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.
  • amino acid residues it would be understood that an alanine residue in a polypeptide or peptide refers to the presence of a residue having a structure given by the formula:
  • Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance.
  • the disclosed compounds can be isotopically-labeled or isotopically- substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 14 C, 15 N, 18 0, 17 0, 35 S, 18 F, and 36 Cl, respectively.
  • Compounds further comprise prodrugs thereof and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention.
  • Certain isotopically-labeled compounds of the present invention for example those into which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-l4, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability.
  • isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.
  • the compounds described in the invention can be present as a solvate.
  • the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate.
  • the compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution.
  • one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates.
  • the invention includes all such possible solvates.
  • co-crystal means a physical association of two or more molecules which owe their stability through non-covalent interaction.
  • One or more components of this molecular complex provide a stable framework in the crystalline lattice.
  • the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g.“Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et al., The Royal Society of Chemistry, 1889-1896, 2004.
  • Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.
  • administering can refer to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intraosseous, intraocular, intracranial, intraperitoneal, intralesional, intranasal, intracardiac, intraarticular, intracavernous, intrathecal, intravireal, intracerebral, and intracerebroventricular, intratympanic, intracochlear, rectal, vaginal, by inhalation, by catheters, stents or via an implanted reservoir or other device that administers, either actively or passively (e.g.
  • a composition the perivascular space and adventitia can contain a composition or formulation disposed on its surface, which can then dissolve or be otherwise distributed to the surrounding tissue and cells.
  • the term“parenteral” can include subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
  • therapeutic agent can refer to any substance, compound, molecule, and the like, which can be biologically active or otherwise can induce a pharmacologic, immunogenic, biologic and/or physiologic effect on a subject to which it is administered to by local and/or systemic action.
  • a therapeutic agent can be a primary active agent, or in other words, the component(s) of a composition to which the whole or part of the effect of the composition is attributed.
  • a therapeutic agent can be a secondary therapeutic agent, or in other words, the component s) of a composition to which an additional part and/or other effect of the composition is attributed.
  • the term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like.
  • therapeutic agents are described in well-known literature references such as the Merck Index (l4th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (l2th edition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.
  • the term“therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, an
  • the agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas.
  • therapeutic agent also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro- drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
  • kit means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
  • instruction(s) means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.
  • Non-covalent interactions can include ionic bonds, electrostatic interactions, van der Walls forces, dipole-dipole interactions, dipole-induced-dipole interactions, London dispersion forces, hydrogen bonding, halogen bonding, electromagnetic interactions, p-p interactions, cation-p interactions, anion-p interactions, polar p-interactions, and hydrophobic effects.
  • subject can refer to a vertebrate organism, such as a mammal (e.g. human).
  • Subject can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.
  • the terms “treating” and “treatment” can refer generally to obtaining a desired pharmacological and/or physiological effect.
  • the effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as a microbial infection or a microbial infection involving an antibiotic resistant microbe, e.g., antibiotic resistant Staphylococcus aureus and/or colistin-resistant strains of Acinetobacter baumannii.
  • the effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition.
  • treatment can include any treatment of a microbial infection in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions.
  • treatment as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment.
  • Those in need of treatment can include those already with the disorder and/or those in which the disorder is to be prevented.
  • treating can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition.
  • Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.
  • dose can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.
  • “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.
  • an effective amount can refer to the amount of a disclosed compound or pharmaceutical composition provided herein that is sufficient to effect beneficial or desired biological, emotional, medical, or clinical response of a cell, tissue, system, animal, or human.
  • An effective amount can be administered in one or more administrations, applications, or dosages.
  • the term can also include within its scope amounts effective to enhance or restore to substantially normal physiological function.
  • the term“therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts.
  • the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
  • the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • a response to a therapeutically effective dose of a disclosed compound and/or pharmaceutical composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • the amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • prophylactically effective amount refers to an amount effective for preventing onset or initiation of a disease or condition.
  • prevent refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
  • the term“pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
  • pharmaceutically acceptable salts means salts of the active principal agents which are prepared with acids or bases that are tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include, but are not limited to; sodium, potassium, calcium, ammonium, organic amino, magnesium salt, lithium salt, strontium salt or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable acid addition salts include, but are not limited to; those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolyl sulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as argin, argon, n, n
  • esters of compounds of the present disclosure which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • examples of pharmaceutically acceptable, non- toxic esters of the present disclosure include C 1 -to-C 6 alkyl esters and C 5 -to-C 7 cycloalkyl esters, although C 1 -to-C 4 alkyl esters are preferred.
  • Esters of disclosed compounds can be prepared according to conventional methods.
  • esters can be appended onto hydroxy groups by reaction of the compound that contains the hydroxy group with acid and an alkylcarboxylic acid such as acetic acid, or with acid and an arylcarboxylic acid such as benzoic acid.
  • the pharmaceutically acceptable esters are prepared from compounds containing the carboxylic acid groups by reaction of the compound with base such as triethylamine and an alkyl halide, for example with methyl iodide, benzyl iodide, cyclopentyl iodide or alkyl triflate. They also can be prepared by reaction of the compound with an acid such as hydrochloric acid and an alcohol such as ethanol or methanol.
  • the term“pharmaceutically acceptable amide” refers to non-toxic amides of the present disclosure derived from ammonia, primary C 1 -to-C 6 alkyl amines and secondary C 1 -to-C 6 dialkyl amines. In the case of secondary amines, the amine can also be in the form of a 5- or 6- membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C 1 -to-C 3 alkyl primary amides and C 1 -to-C 2 dialkyl secondary amides are preferred. Amides of disclosed compounds can be prepared according to conventional methods.
  • Pharmaceutically acceptable amides can be prepared from compounds containing primary or secondary amine groups by reaction of the compound that contains the amino group with an alkyl anhydride, aryl anhydride, acyl halide, or aroyl halide.
  • the pharmaceutically acceptable amides are prepared from compounds containing the carboxylic acid groups by reaction of the compound with base such as triethylamine, a dehydrating agent such as dicyclohexyl carbodiimide or carbonyl diimidazole, and an alkyl amine, dialkylamine, for example with methylamine, diethylamine, and piperidine.
  • compositions can contain a compound of the present disclosure in the form of a pharmaceutically acceptable prodrug.
  • prodrug represents those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
  • Prodrugs of the present disclosure can be rapidly transformed in vivo to a parent compound having a structure of a disclosed compound, for example, by hydrolysis in blood.
  • a thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).
  • the term“derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g ., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.
  • exemplary derivatives include salts, esters, and amides, salts of esters or amides, and N-oxides of a parent compound.
  • contacting refers to bringing a disclosed compound or pharmaceutical composition in proximity to a cell, a target protein, or other biological entity together in such a manner that the disclosed compound or pharmaceutical composition can affect the activity of the a cell, target protein, or other biological entity, either directly; i.e., by interacting with the cell, target protein, or other biological entity itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the cell, target protein, or other biological entity itself is dependent.
  • Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art.
  • the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St.
  • compositions of the invention Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary.
  • temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
  • the phage panning against intact cells is remarkably convenient and powerful, allowing facile incorporation of negative screens and internal competitors.
  • abundant endogenous protein could compete for iminoboronate conjugation, thereby inhibiting the bacterial binding of an APB A-containing peptide.
  • the data presented in the present disclosure unequivocally show that this protein interference problem can be overcome by including serum albumin in the screening mixture and the reversible covalent binding mechanism can afford highly selective binders in complex biological milieu.
  • the APBA dimer library can be extended to discovering binders of various bacterial pathogens. This is feasible given that iminoboronate chemistry is generally applicable to primary amines, which can be abundant on bacterial cells particularly those showing antibiotic resistance. Amino acid modification of phospholipids, such as Lys-PG synthesis in S. aureus , have been documented for a number of bacterial species as a resistance mechanism to host defense peptides and neutrophil clearance. Such lipid modification affords a high abundance of amino groups (a- and e-amine of lysine) on a bacterial surface. Similarly, alanylation of teichoic acids results in abundant alanyl ester structures with a-amines available for iminoboronate conjugation.
  • LPS lipopolysaccharide
  • LOS lipooligosaccharide
  • the present disclosure provides that a bacterial binder identified from phage display can be readily converted to targeted antibiotics that specifically eradicate the corresponding strain of bacteria.
  • the facile generation of targeted antibiotics is of contemporary importance given the undesirable consequences of broad-spectrum antibiotics, which inevitably cultivate antibiotic resistance and cause damage to human microbiota.
  • the present disclosure utilizes a phototoxin as the bactericidal agent, it is conceivable that antibiotics of other modes of action, such as those targeting cell membranes including vancomycin and daptomycin, can be utilized to build peptide-antibiotic conjugates, which will expand the scope of our strategy to create targeted antibiotics.
  • the phage display platform can be further developed to include additional phage libraries with reversible covalent warheads. This can be accomplished by varying the designs of the reversible covalent warheads and introducing crosslinks to the linear peptide architecture to generate cyclic and multicyclic peptides. Additional phage libraries as such can maximize the chance of success for a diverse range of bacterial pathogens.
  • the design principles of peptide- antibiotic conjugates that can be used for the selective clearance of a pathogenic bacteria can also be systematically studies.
  • the disclosed phage display libraries comprise an APBA residue.
  • the disclosed phage display libraries comprise two APBA modified cysteine residues in a peptide expressed on a phage particle surface.
  • an APBA residue has a structure given by the following formula:
  • APB A residue is a structure given by the following formula:
  • a disclosed phage display library comprises peptide structures on an external surface of a phage particle comprising two cysteine residues having an APBA residue as shown herein above.
  • a disclosed phage display library comprises an APBA modified peptide, i.e., peptides expressed on an external surface of a phage particle such that the peptides comprise an APBA modified cysteine residue, as discussed above,
  • a disclosed phage display library can be prepared by chemically modifying an APBA modifiable phage display library, e.g., using an APBA-IA reagent.
  • an APBA-IA reagent is a compound comprising an APBA moiety and an iodoacetamide residue having a structure given by the following formula:
  • a 1 and A 2 are independently a C1-C6 alkyl.
  • a particular example of APBA-IA is (2-acetyl-5-(3-(2-iodoacetamido)propoxy)phenyl)boronic acid, that is, a compound having a structure given by the following formula:
  • a disclosed APBA modifiable dimer phage library is a phage display library comprising a peptide sequence on an external portion of a phage particle as follows:
  • a disclosed APBA modifiable dimer phage library comprises APBA modifiable peptides on the surface of a phage particle have the structure given by the following formula:
  • R 1 , R 2 , and R 3 are independently selected from a moiety that is an amino acid side chain except a cysteine side chain; wherein n is an integer selected from 5, 6, 7, 8, 9, and 10; and wherein m is an integer selected from 1, 2, 3, 4, and 5.
  • an APBA modifiable dimer phage library is a phage display library comprising a peptide sequence on an external portion of a phage particle as follows:
  • an APBA modifiable dimer phage library comprises APBA modifiable peptides on the surface of a phage particle have the structure given by the following formula: wherein each occurrence of R 2 is independently selected from a moiety that is an amino acid side chain except cysteine; wherein n is an integer selected from 5, 6, 7, 8, 9, and 10; and wherein m is an integer selected from 1, 2, 3, 4, and 5.
  • a disclosed phage display library is an APBA dimer phage library.
  • a exemplary phage display library comprises peptide sequences on an external portion of a phage particle as follows:
  • C* indicates a cysteine residue modified to comprise an APBA residue
  • each instance of X is an amino acid independently selected from D, E, K, R, H, Y, N, Q, S, T, G, A, V, L, I, M, P, F, and W
  • n is an integer selected from 5, 6, 7, 8, 9, and 10
  • m is an integer selected from 1, 2, 3, 4, and 5.
  • a dislcosed APBA dimer phage library comprises APBA modified peptides on the surface of a phage particle having a structure given by the following formula:
  • each of A 1 and A 2 are independently a C1-C6 alkyl; wherein each occurrence of R 1 , R 2 , and R 3 are independently selected from a moiety that is an amino acid side chain except cysteine; wherein n is an integer selected from 5, 6, 7, 8, 9, and 10; and wherein m is an integer selected from 1, 2, 3, 4, and 5.
  • an APBA dimer phage library comprises modified peptides on the surface of a phage particle having a structure given by the following formula:
  • a disclosed APBA dimer phage library is a phage display library comprising peptide sequence on an external portion of a phage particle as follows:
  • an APBA dimer phage library comprises APBA modified peptides on the surface of a phage particle having a structure given by the following formula:
  • an APBA dimer phage library comprises APBA modified peptides on the surface of a phage particle having a structure given by the following formula:
  • the present disclosure pertains to therapeutic APBA peptides. That is, APBA modified peptides which are understood to be peptides comprising two APBA modified cysteine residues.
  • An exemplary disclosed therapeutic APBA peptide is a peptide sequence as follows:
  • XC*(X)nC*(X)m wherein C* indicates a cysteine residue modified to comprise an APBA residue; wherein each instance of X is an amino acid independently selected from D, E, K, R, H, Y, N, Q, S, T, G, A, V, L, I, M, P, F, and W; wherein n is an integer selected from 5, 6, 7, 8, 9, and 10; wherein m is an integer selected from 1, 2, 3, 4, and 5.
  • a disclosed therapeutic APBA peptide is a peptide having a structure given by the formula:
  • each of A 1 and A 2 are independently a C1-C6 alkyl; wherein each occurrence of R 1 , R 2 , and R 3 are independently selected from a side chain of an amino acid selected from D, E, K, R, H, Y, N, Q, S, T, G, A, V, L, I, M, P, F, and W; wherein each of R 10 and R 20 is selected from hydrogen, an antibiotic residue, a phototoxin residue, and a detectable label residue; wherein n is an integer selected from 5, 6, 7, 8, 9, and 10; and wherein m is an integer selected from 1, 2, 3, 4, and 5.
  • a disclosed therapeutic APBA peptide is a peptide having a structure given by the formula:
  • a disclosed therapeutic APBA peptide is a peptide having a structure given by the formula:
  • a disclosed therapeutic APBA peptide is a peptide having a structure given by the formula:
  • the present disclosure relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one therapeutic APBA peptide, or a pharmaceutically acceptable salt thereof.
  • pharmaceutically-acceptable carriers means one or more of a pharmaceutically acceptable diluents, preservatives, antioxidants, solubilizers, emulsifiers, coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, and adjuvants.
  • the disclosed pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy and pharmaceutical sciences.
  • the disclosed pharmaceutical compositions comprise a therapeutically effective amount of at least one disclosed therapeutic APBA peptide, optionally one or more other therapeutic agent, and optionally one or more adjuvant.
  • the disclosed pharmaceutical compositions include those suitable for oral, rectal, topical, pulmonary, nasal, and parenteral administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered.
  • the disclosed pharmaceutical composition can be formulated to allow administration orally, nasally, via inhalation, parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally.
  • parenteral administration includes administration by bolus injection or infusion, as well as administration by intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, and at least one disclosed therapeutic APBA peptide.
  • at least one disclosed therapeutic APBA peptide may be formulated into various pharmaceutical forms for administration purposes.
  • salts can be prepared from pharmaceutically acceptable non toxic bases or acids.
  • salts of the disclosed therapeutic APBA peptide are those wherein the counter ion is pharmaceutically acceptable.
  • salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not, are contemplated by the present disclosure.
  • Pharmaceutically acceptable acid and base addition salts are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the disclosed compounds are able to form.
  • a disclosed therapeutic APBA peptide comprising an acidic group or moiety, e.g., a carboxylic acid group
  • a pharmaceutically acceptable salt can be used to prepare a pharmaceutically acceptable salt.
  • a disclosed compound may comprise an isolation step comprising treatment with a suitable inorganic or organic base.
  • base addition salts can be readily prepared using conventional techniques, e.g., by treating the corresponding acidic compounds with an aqueous solution containing the desired pharmacologically acceptable cations and then evaporating the resulting solution to dryness, preferably under reduced pressure.
  • they also can be prepared by mixing lower alkanolic solutions of the acidic compounds and the desired alkali metal alkoxide together, and then evaporating the resulting solution to dryness in the same manner as before.
  • Bases which can be used to prepare the pharmaceutically acceptable base-addition salts of the base compounds are those which can form non-toxic base-addition salts, i.e., salts containing pharmacologically acceptable cations such as, alkali metal cations (e.g., lithium, potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water- soluble amine addition salts such as N-methylglucamine-(meglumine), lower alkanolammonium and other such bases of organic amines.
  • pharmacologically acceptable cations such as, alkali metal cations (e.g., lithium, potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water- soluble amine addition salts such as N-methylglucamine-(meglumine), lower alkanolammonium and other such bases of organic amines.
  • derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines.
  • such pharmaceutically acceptable organic non-toxic bases include, but are not limited to, ammonia, methylamine, ethylamine, propylamine, isopropylamine, any of the four butylamine isomers, betaine, caffeine, choline, dimethylamine, di ethylamine, diethanolamine, dipropylamine, diisopropylamine, di-//-butylamine, N,N'-dibenzylethylenediamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, tromethamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, quinuclidine, pyridine,
  • a disclosed therapeutic APBA peptide comprising a protonatable group or moiety, e.g., an amino group
  • a pharmaceutically acceptable salt can be used to prepare a pharmaceutically acceptable salt.
  • such a disclosed compound may comprise an isolation step comprising treatment with a suitable inorganic or organic acid.
  • acid addition salts can be readily prepared using conventional techniques, e.g., by treating the corresponding basic compounds with an aqueous solution containing the desired pharmacologically acceptable anions and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, they also can be prepared by treating the free base form of the disclosed compound with a suitable pharmaceutically acceptable non-toxic inorganic or organic acid.
  • Acids which can be used to prepare the pharmaceutically acceptable acid-addition salts of the base compounds are those which can form non-toxic acid-addition salts, i.e., salts containing pharmacologically acceptable anions formed from their corresponding inorganic and organic acids.
  • non-toxic acid-addition salts i.e., salts containing pharmacologically acceptable anions formed from their corresponding inorganic and organic acids.
  • inorganic acids include hydrochloric hydrobromic, sulfuric, nitric, phosphoric and the like.
  • organic acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, isethionic, lactic, maleic, malic, mandelicmethanesulfonic, mucic, pamoic, pantothenic, succinic, tartaric, p-toluenesulfonic acid and the like.
  • the acid-addition salt comprises an anion formed from hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.
  • the therapeutic APBA peptides of the present disclosure, or pharmaceutically acceptable salts thereof, of the present disclosure can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
  • the pharmaceutical compositions of the present disclosure can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient.
  • compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion.
  • the compounds of the present disclosure, and/or pharmaceutically acceptable salt(s) thereof can also be administered by controlled release means and/or delivery devices.
  • the compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.
  • unit dosage form refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. That is, a“unit dosage form” is taken to mean a single dose wherein all active and inactive ingredients are combined in a suitable system, such that the patient or person administering the drug to the patient can open a single container or package with the entire dose contained therein, and does not have to mix any components together from two or more containers or packages.
  • unit dosage forms are tablets (including scored or coated tablets), capsules or pills for oral administration; single dose vials for injectable solutions or suspension; suppositories for rectal administration; powder packets; wafers; and segregated multiples thereof.
  • This list of unit dosage forms is not intended to be limiting in any way, but merely to represent typical examples of unit dosage forms.
  • compositions disclosed herein comprise a therapeutic APBA peptide of the present disclosure (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents.
  • the disclosed pharmaceutical compositions can include a pharmaceutically acceptable carrier and a disclosed compound, or a pharmaceutically acceptable salt thereof.
  • a disclosed compound, or pharmaceutically acceptable salt thereof can also be included in a pharmaceutical composition in combination with one or more other therapeutically active compounds.
  • compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered.
  • the pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
  • the therapeutic APBA peptides described herein are typically to be administered in admixture with suitable pharmaceutical diluents, excipients, extenders, or carriers (termed herein as a pharmaceutically acceptable carrier, or a carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • suitable pharmaceutical diluents, excipients, extenders, or carriers suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the deliverable compound will be in a form suitable for oral, rectal, topical, intravenous injection or parenteral administration.
  • Carriers include solids or liquids, and the type of carrier is chosen based on the type of administration being used.
  • the compounds may be administered as a dosage that has a known quantity of the compound.
  • oral administration can be a preferred dosage form, and tablets and capsules represent the most advantageous oral dosage unit forms in which case solid pharmaceutical carriers are obviously employed.
  • other dosage forms may be suitable depending upon clinical population (e.g., age and severity of clinical condition), solubility properties of the specific disclosed compound used, and the like.
  • the disclosed compounds can be used in oral dosage forms such as pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • any convenient pharmaceutical media can be employed.
  • oral liquid preparations such as suspensions, elixirs and solutions
  • carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like
  • oral solid preparations such as powders, capsules and tablets.
  • tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed.
  • tablets can be coated by standard aqueous or nonaqueous techniques.
  • compositions in an oral dosage form can comprise one or more pharmaceutical excipient and/or additive.
  • suitable excipients and additives include gelatin, natural sugars such as raw sugar or lactose, lecithin, pectin, starches (for example com starch or amylose), dextran, polyvinyl pyrrolidone, polyvinyl acetate, gum arabic, alginic acid, tylose, talcum, lycopodium, silica gel (for example colloidal), cellulose, cellulose derivatives (for example cellulose ethers in which the cellulose hydroxy groups are partially etherified with lower saturated aliphatic alcohols and/or lower saturated, aliphatic oxyalcohols, for example methyl oxypropyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate), fatty acids as well as magnesium, calcium or aluminum salts of fatty acids with 12 to 22
  • auxiliary substances useful in preparing an oral dosage form are those which cause disintegration (so-called disintegrants), such as: cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, sodium carboxymethyl cellulose or microcrystalline cellulose.
  • Conventional coating substances may also be used to produce the oral dosage form.
  • Plasticizing agents that may be considered as coating substances in the disclosed oral dosage forms are: citric and tartaric acid esters (acetyl -tri ethyl citrate, acetyl tributyl-, tributyl-, tri ethyl -citrate); glycerol and glycerol esters (glycerol diacetate, -triacetate, acetylated monoglycerides, castor oil); phthalic acid esters (dibutyl-, diamyl-, diethyl-, dimethyl-, dipropyl- phthalate), di-(2-methoxy- or 2-ethoxy ethyl)-phthalate, ethylphthalyl glycolate, butylphthalylethyl glycolate and butylglycolate; alcohols (propylene glycol, polyethylene glycol of various chain lengths), adipates (diethyladipate, di-(2-methoxy- or
  • suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents may be included as carriers.
  • the pharmaceutical carrier employed can be, for example, a solid, liquid, or gas.
  • solid carriers include, but are not limited to, lactose, terra alba, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol, sorbitol talc, starch, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid.
  • liquid carriers are sugar syrup, peanut oil, olive oil, and water.
  • gaseous carriers include carbon dioxide and nitrogen.
  • a binder can include, for example, starch, gelatin, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • a disintegrator can include, for example, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • an oral dosage form such as a solid dosage form, can comprise a disclosed compound that is attached to polymers as targetable drug carriers or as a prodrug.
  • Suitable biodegradable polymers useful in achieving controlled release of a drug include, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, caprolactones, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and hydrogels, preferably covalently crosslinked hydrogels.
  • Tablets may contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a tablet containing a disclosed compound can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants.
  • Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
  • a solid oral dosage form such as a tablet
  • enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid- methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate.
  • enteric coating materials may be selected on the basis of testing to achieve an enteric coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength (e.g., see S. C. Porter et al.“The Properties of Enteric Tablet Coatings Made From Polyvinyl Acetate-phthalate and Cellulose acetate Phthalate”, J. Pharm. Pharmacol. 22:42p (1970)).
  • the enteric coating may comprise hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate- phthalate and cellulose acetate phthalate.
  • an oral dosage form can be a solid dispersion with a water soluble or a water insoluble carrier.
  • water soluble or water insoluble carrier include, but are not limited to, polyethylene glycol, polyvinylpyrrolidone, hydroxypropylmethyl-cellulose, phosphatidylcholine, polyoxyethylene hydrogenated castor oil, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, or hydroxypropylmethylcellulose, ethyl cellulose, or stearic acid.
  • an oral dosage form can be in a liquid dosage form, including those that are ingested, or alternatively, administered as a mouth wash or gargle.
  • a liquid dosage form can include aqueous suspensions, which contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain various excipients.
  • the pharmaceutical compositions of the present disclosure may also be in the form of oil-in-water emulsions, which may also contain excipients such as sweetening and flavoring agents.
  • water particularly sterile water, or physiologically acceptable organic solvents, such as alcohols (ethanol, propanol, isopropanol, 1, 2-propylene glycol, polyglycols and their derivatives, fatty alcohols, partial esters of glycerol), oils (for example peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovine hoof oil), paraffins, dimethyl sulphoxide, triglycerides and the like.
  • alcohols ethanol, propanol, isopropanol, 1, 2-propylene glycol, polyglycols and their derivatives, fatty alcohols, partial esters of glycerol
  • oils for example peanut oil, olive oil, sesame oil, almond oil, sunflower oil, soya bean oil, castor oil, bovine hoof oil
  • paraffins dimethyl sulphoxide, triglycerides and the like.
  • a liquid dosage form such as a drinkable solutions
  • the following substances may be used as stabilizers or solubilizers: lower aliphatic mono- and multivalent alcohols with 2- 4 carbon atoms, such as ethanol, n-propanol, glycerol, polyethylene glycols with molecular weights between 200-600 (for example 1 to 40% aqueous solution), diethylene glycol monoethyl ether, 1, 2-propylene glycol, organic amides, for example amides of aliphatic Cl-C6-carboxylic acids with ammonia or primary, secondary or tertiary Cl-C4-amines or Cl-C4-hydroxy amines such as urea, urethane, acetamide, N-methyl acetamide, N,N-diethyl acetamide, N,N-dimethyl acetamide, lower aliphatic amines and diamines with 2-6 carbon atoms, such as ethylene diamine
  • solubilizers and emulsifiers such as the following non-limiting examples can be used: polyvinyl pyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phosphatides such as lecithin, acacia, tragacanth, polyoxyethylated sorbitan monooleate and other ethoxylated fatty acid esters of sorbitan, polyoxyethylated fats, polyoxyethylated oleotriglycerides, linolizated oleotriglycerides, polyethylene oxide condensation products of fatty alcohols, alkylphenols or fatty acids or also 1- methyl-3-(2-hydroxyethyl)imidazolidone-(2).
  • solubilizers and emulsifiers such as the following non-limiting examples can be used: polyvinyl pyrrolidone, sorbitan fatty acid esters such as sorbitan trioleate, phosphatides
  • polyoxyethylated means that the substances in question contain polyoxyethylene chains, the degree of polymerization of which generally lies between 2 and 40 and in particular between 10 and 20.
  • Polyoxyethylated substances of this kind may for example be obtained by reaction of hydroxyl group-containing compounds (for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid radicals) with ethylene oxide (for example 40 Mol ethylene oxide per 1 Mol glyceride).
  • hydroxyl group-containing compounds for example mono- or diglycerides or unsaturated compounds such as those containing oleic acid radicals
  • ethylene oxide for example 40 Mol ethylene oxide per 1 Mol glyceride
  • oleotriglycerides are olive oil, peanut oil, castor oil, sesame oil, cottonseed oil, com oil. See also Dr. H. P. Fiedler“Lexikon der Hillsstoffe fiir Pharmazie, Kostnetik und angrenzende füre” 1971, pages 191-
  • a liquid dosage form can further comprise preservatives, stabilizers, buffer substances, flavor correcting agents, sweeteners, colorants, antioxidants and complex formers and the like.
  • Complex formers which may be for example be considered are: chelate formers such as ethylene diamine retrascetic acid, nitrilotriacetic acid, diethylene triamine pentacetic acid and their salts.
  • a liquid dosage form with physiologically acceptable bases or buffers may optionally be necessary to stabilize a liquid dosage form with physiologically acceptable bases or buffers to a pH range of approximately 6 to 9. Preference may be given to as neutral or weakly basic a pH value as possible (up to pH 8).
  • a parenteral injection form or an intravenous injectable form
  • co-solvents such as alcohols may improve the solubility and/or the stability of the compounds according to the present disclosure in pharmaceutical compositions.
  • a disclosed liquid dosage form, a parenteral injection form, or an intravenous injectable form can further comprise liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles.
  • liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • compositions of the present disclosure suitable injection, such as parenteral administration, such as intravenous, intramuscular, or subcutaneous administration.
  • Pharmaceutical compositions for injection can be prepared as solutions or suspensions of the active compounds in water.
  • a suitable surfactant can be included such as, for example, hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
  • compositions of the present disclosure suitable for parenteral administration can include sterile aqueous or oleaginous solutions, suspensions, or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some aspects, the final injectable form is sterile and must be effectively fluid for use in a syringe.
  • the pharmaceutical compositions should be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol ( e.g ., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
  • Injectable solutions for example, can be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed.
  • a disclosed parenteral formulation can comprise about 0.01-0.1 M, e.g. about 0.05 M, phosphate buffer. In a further aspect, a disclosed parenteral formulation can comprise about 0.9% saline.
  • a disclosed parenteral pharmaceutical composition can comprise pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions.
  • pharmaceutically acceptable carriers such as aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include but not limited to water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.
  • a disclosed parenteral pharmaceutical composition can comprise may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • Also contemplated for injectable pharmaceutical compositions are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the subject or patient.
  • the disclosed compounds can also be formulated as a depot preparation.
  • Such long acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.
  • compositions of the present disclosure can be in a form suitable for topical administration.
  • topical application means administration onto a biological surface, whereby the biological surface includes, for example, a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas) or a mucosal membrane.
  • a skin area e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas
  • a mucosal membrane e.g., a skin area (e.g., hands, forearms, elbows, legs, face, nails, anus and genital areas) or a mucosal membrane.
  • a topical pharmaceutical composition can be in a form of a cream, an ointment, a paste, a gel, a lotion, milk, a suspension, an aerosol, a spray, foam, a dusting powder, a pad, and a patch. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the present disclosure, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.
  • the carrier optionally comprises a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
  • These compositions may be administered in various ways, e.g., as a transdermal patch, as a spot-on, as an ointment.
  • Ointments are semisolid preparations, typically based on petrolatum or petroleum derivatives.
  • the specific ointment base to be used is one that provides for optimum delivery for the active agent chosen for a given formulation, and, preferably, provides for other desired characteristics as well (e.g., emollience).
  • an ointment base should be inert, stable, nonirritating and nonsensitizing. As explained in Remington: The Science and Practice of Pharmacy, l9th Ed., Easton, Pa. : Mack Publishing Co. (1995), pp.
  • ointment bases may be grouped in four classes: oleaginous bases; emulsifiable bases; emulsion bases; and water-soluble bases.
  • Oleaginous ointment bases include, for example, vegetable oils, fats obtained from animals, and semisolid hydrocarbons obtained from petroleum.
  • Emulsifiable ointment bases also known as absorbent ointment bases, contain little or no water and include, for example, hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.
  • Emulsion ointment bases are either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and include, for example, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.
  • W/O water-in-oil
  • O/W oil-in-water
  • Preferred water-soluble ointment bases are prepared from polyethylene glycols of varying molecular weight.
  • Lotions are preparations that are to be applied to the skin surface without friction. Lotions are typically liquid or semiliquid preparations in which solid particles, including the active agent, are present in a water or alcohol base. Lotions are typically preferred for treating large body areas, due to the ease of applying a more fluid composition. Lotions are typically suspensions of solids, and oftentimes comprise a liquid oily emulsion of the oil-in-water type. It is generally necessary that the insoluble matter in a lotion be finely divided. Lotions typically contain suspending agents to produce better dispersions as well as compounds useful for localizing and holding the active agent in contact with the skin, such as methylcellulose, sodium carboxymethyl-cellulose, and the like.
  • Creams are viscous liquids or semisolid emulsions, either oil-in-water or water-in-oil.
  • Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase.
  • the oil phase also called the“internal” phase, is generally comprised of petrolatum and/or a fatty alcohol such as cetyl or stearyl alcohol.
  • the aqueous phase typically, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. Reference may be made to Remington: The Science and Practice of Pharmacy, supra, for further information.
  • Pastes are semisolid dosage forms in which the bioactive agent is suspended in a suitable base. Depending on the nature of the base, pastes are divided between fatty pastes or those made from a single-phase aqueous gel.
  • the base in a fatty paste is generally petrolatum, hydrophilic petrolatum and the like.
  • the pastes made from single-phase aqueous gels generally incorporate carboxymethylcellulose or the like as a base. Additional reference may be made to Remington: The Science and Practice of Pharmacy, for further information.
  • Gel formulations are semisolid, suspension-type systems.
  • Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the carrier liquid, which is typically aqueous, but also, preferably, contain an alcohol and, optionally, an oil.
  • Preferred organic macromolecules, i.e., gelling agents are crosslinked acrylic acid polymers such as the family of carbomer polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the trademark CarbopolTM.
  • hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinylalcohol; modified cellulose, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin.
  • dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing or stirring, or combinations thereof.
  • Sprays generally provide the active agent in an aqueous and/or alcoholic solution which can be misted onto the skin for delivery.
  • Such sprays include those formulated to provide for concentration of the active agent solution at the site of administration following delivery, e.g., the spray solution can be primarily composed of alcohol or other like volatile liquid in which the active agent can be dissolved.
  • the carrier evaporates, leaving concentrated active agent at the site of administration.
  • Foam compositions are typically formulated in a single or multiple phase liquid form and housed in a suitable container, optionally together with a propellant which facilitates the expulsion of the composition from the container, thus transforming it into a foam upon application.
  • Other foam forming techniques include, for example the “Bag-in-a-can” formulation technique.
  • Compositions thus formulated typically contain a low-boiling hydrocarbon, e.g., isopropane. Application and agitation of such a composition at the body temperature cause the isopropane to vaporize and generate the foam, in a manner similar to a pressurized aerosol foaming system.
  • Foams can be water-based or aqueous alkanolic, but are typically formulated with high alcohol content which, upon application to the skin of a user, quickly evaporates, driving the active ingredient through the upper skin layers to the site of treatment.
  • Skin patches typically comprise a backing, to which a reservoir containing the active agent is attached.
  • the reservoir can be, for example, a pad in which the active agent or composition is dispersed or soaked, or a liquid reservoir.
  • Patches typically further include a frontal water permeable adhesive, which adheres and secures the device to the treated region. Silicone rubbers with self-adhesiveness can alternatively be used. In both cases, a protective permeable layer can be used to protect the adhesive side of the patch prior to its use.
  • Skin patches may further comprise a removable cover, which serves for protecting it upon storage.
  • Examples of patch configuration which can be utilized with the present invention include a single-layer or multi-layer drug-in-adhesive systems which are characterized by the inclusion of the drug directly within the skin-contacting adhesive.
  • the adhesive not only serves to affix the patch to the skin, but also serves as the formulation foundation, containing the drug and all the excipients under a single backing film.
  • a membrane is disposed between two distinct drug-in-adhesive layers or multiple drug-in-adhesive layers are incorporated under a single backing film.
  • Examples of pharmaceutically acceptable carriers that are suitable for pharmaceutical compositions for topical applications include carrier materials that are well-known for use in the cosmetic and medical arts as bases for e.g., emulsions, creams, aqueous solutions, oils, ointments, pastes, gels, lotions, milks, foams, suspensions, aerosols and the like, depending on the final form of the composition.
  • suitable carriers according to the present invention therefore include, without limitation, water, liquid alcohols, liquid glycols, liquid polyalkylene glycols, liquid esters, liquid amides, liquid protein hydrolysates, liquid alkylated protein hydrolysates, liquid lanolin and lanolin derivatives, and like materials commonly employed in cosmetic and medicinal compositions.
  • suitable carriers include, without limitation, alcohols, such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannitol, and propylene glycol; ethers such as diethyl or dipropyl ether; polyethylene glycols and methoxypolyoxyethylenes (carbowaxes having molecular weight ranging from 200 to 20,000); polyoxyethylene glycerols, polyoxyethylene sorbitols, stearoyl diacetin, and the like.
  • alcohols such as, for example, monohydric and polyhydric alcohols, e.g., ethanol, isopropanol, glycerol, sorbitol, 2-methoxyethanol, diethyleneglycol, ethylene glycol, hexyleneglycol, mannito
  • Topical compositions of the present disclosure can, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the dispenser device may, for example, comprise a tube.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • Compositions comprising the topical composition of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Another patch system configuration which can be used by the present invention is a reservoir transdermal system design which is characterized by the inclusion of a liquid compartment containing a drug solution or suspension separated from the release liner by a semi- permeable membrane and adhesive.
  • the adhesive component of this patch system can either be incorporated as a continuous layer between the membrane and the release liner or in a concentric configuration around the membrane.
  • Yet another patch system configuration which can be utilized by the present invention is a matrix system design which is characterized by the inclusion of a semisolid matrix containing a drug solution or suspension which is in direct contact with the release liner.
  • the component responsible for skin adhesion is incorporated in an overlay and forms a concentric configuration around the semisolid matrix.
  • compositions of the present disclosure can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
  • compositions containing a compound of the present disclosure, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.
  • the pharmaceutical composition may be packaged in a variety of ways.
  • an article for distribution includes a container that contains the pharmaceutical composition in an appropriate form.
  • Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, foil blister packs, and the like.
  • the container may also include a tamper proof assemblage to prevent indiscreet access to the contents of the package.
  • the container typically has deposited thereon a label that describes the contents of the container and any appropriate warnings or instructions.
  • the disclosed pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • Pharmaceutical compositions comprising a disclosed compound formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • the exact dosage and frequency of administration depends on the particular disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, solvate, or polymorph thereof, a hydrate thereof, a solvate thereof, a polymorph thereof, or a stereochemically isomeric form thereof; the particular condition being treated and the severity of the condition being treated; various factors specific to the medical history of the subject to whom the dosage is administered such as the age; weight, sex, extent of disorder and general physical condition of the particular subject, as well as other medication the individual may be taking; as is well known to those skilled in the art. Furthermore, it is evident that said effective daily amount may be lowered or increased depending on the response of the treated subject and/or depending on the evaluation of the physician prescribing the compounds of the present disclosure.
  • the pharmaceutical composition will comprise from 0.05 to 99 % by weight, preferably from 0.1 to 70 % by weight, more preferably from 0.1 to 50 % by weight of the active ingredient, and, from 1 to 99.95 % by weight, preferably from 30 to 99.9 % by weight, more preferably from 50 to 99.9 % by weight of a pharmaceutically acceptable carrier, all percentages being based on the total weight of the composition.
  • an appropriate dosage level will generally be about 0.01 to 1000 mg per kg patient body weight per day and can be administered in single or multiple doses.
  • the dosage level will be about 0.1 to about 500 mg/kg per day, about 0.1 to 250 mg/kg per day, or about 0.5 to 100 mg/kg per day.
  • a suitable dosage level can be about 0.01 to 1000 mg/kg per day, about 0.01 to 500 mg/kg per day, about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day.
  • compositions are preferably provided in the form of tablets containing 1.0 to 1000 mg of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 mg of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated.
  • the compound can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response.
  • Such unit doses as described hereinabove and hereinafter can be administered more than once a day, for example, 2, 3, 4, 5 or 6 times a day.
  • such unit doses can be administered 1 or 2 times per day, so that the total dosage for a 70 kg adult is in the range of 0.001 to about 15 mg per kg weight of subject per administration.
  • dosage is 0.01 to about 1.5 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years.
  • the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs that have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
  • a typical dosage can be one 1 mg to about 100 mg tablet or 1 mg to about 300 mg taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient.
  • the time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
  • the present disclosure is further directed to a method for the manufacture of a medicament for anti-microbial activity (e.g ., treatment of one or more microbial infections) in mammals (e.g., humans) comprising combining one or more disclosed therapeutic APBA peptides with a pharmaceutically acceptable carrier or diluent.
  • a method for manufacturing a medicament comprising combining at least one disclosed therapeutic APBA peptide with a pharmaceutically acceptable carrier or diluent.
  • compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological or clinical conditions.
  • compositions can be prepared from the disclosed therapeutic APBA peptides. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a disclosed therapeutic APBA peptide, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Additionally, the present disclosure relates to a process for preparing such a pharmaceutical composition, characterized in that a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of a compound according to the present disclosure.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a disclosed compound, a product of a disclosed method of making, a pharmaceutically acceptable salt, a hydrate thereof, a solvate thereof, a polymorph thereof, and one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of diseases or conditions for a disclosed compound or the other drugs may have utility as well as to the use of such a composition for the manufacture of a medicament.
  • the present disclosure also relates to a combination of disclosed therapeutic APBA peptide, a pharmaceutically acceptable salt thereof, and a therapeutic agent that is known to treat a microbial infection.
  • the present disclosure also relates to such a combination for use as a medicine.
  • the present disclosure also relates to a product comprising (a) disclosed therapeutic APBA peptide, or a pharmaceutically acceptable salt thereof, and (b) an additional antimicrobial therapeutic agent, as a combined preparation for simultaneous, separate or sequential use in the treatment or prevention of a condition in a mammal, including a human, the treatment or prevention of which is affected or facilitated by the modulatory effect of the disclosed compound and the additional therapeutic agent.
  • the different drugs of such a combination or product may be combined in a single preparation together with pharmaceutically acceptable carriers or diluents, or they may each be present in a separate preparation together with pharmaceutically acceptable carriers or diluents.
  • the present disclosure provides methods of treating an infectious disease comprising administration of a therapeutically effective amount of a disclosed APBA therapeutic peptide or a disclosed pharmaceutical composition to a subject in need thereof.
  • a disclosed APBA therapeutic peptide is inclusive of the disclosed APBA therapeutic peptide, as well as pharmaceutically acceptable salt, hydrate, solvate, or polymorph forms thereof and a disclosed APBA therapeutic peptide further comprising an an antibiotic residue, a phototoxin residue, and/or a detectable label residue
  • reference to a disclosed pharmaceutical composition is inclusive of a pharmaceutical composition comprising a disclosed APBA therapeutic peptide or pharmaceutically acceptable salt, hydrate, solvate, or polymorph forms of a disclosed APBA therapeutic peptide, and pharmaceutical compositions comprising a disclosed APBA therapeutic peptide further comprising an an antibiotic residue, a phototoxin residue, and/or a detectable label residue.
  • a treatment can include binding a disclosed APBA therapeutic peptide, optionally further comprising an antibiotic residue, a phototoxin residue, and/or a detectable label residue, to a target bacteria in a subject infected with said targetbacteria, and wherein the disclosed APBA therapeutic peptide via binding and/or binding with delivery of an an antibiotic residue, a phototoxin residue, and/or a detectable label residue to the target bacteria.
  • a method of treating or preventing a bacterial infection in a subject comprising the step of administering to the subject at least one disclosed APBA therapeutic peptide or at least one disclosed pharmaceutical composition in a dosage and amount effective to treat the disorder in the subject.
  • Also provided is a method for the treatment of one or more disorders associated with infection by a target bacteria wherein inhibiting binding of a disclosed APBA therapeutic peptide can sterilize or decrease the presence of the pathogenic bacteria in a subject comprising the step of administering to the subject at least one disclosed APBA therapeutic peptide or at least one disclosed pharmaceutical composition in a dosage and amount effective to treat the disorder in the subject.
  • Also provided is a method for the treatment of a bacterial infection in a vertebrate animal comprising the step of administering to the mammal at least one disclosed compound, composition, or medicament.
  • the vertebrate animal is a mammal.
  • the vertebrate animal is a fish, a bird, or a mammal.
  • the vertebrate animal is a livestock animal.
  • the vertebrate animal is a companion animal.
  • the vertebrate animal is a farm animal.
  • the vertebrate animal is a zoo animal.
  • the vertebrate animal is a laboratory animal.
  • the vertebrate animal is an aquaculture fish.
  • the vertebrate animal is selected from Bison sp., Bos sp., Canis sp., Capra sp., Equus sp., Felis sp., Callus sp., Lama sp., Meleagris sp., Oryctolagus sp., Ovis sp., and Sus sp.
  • the vertebrate animal has been diagnosed with a need for treatment of the infectious disease prior to the administering step.
  • the disclosure relates to a method for the treatment of an infectious disease in a vertebrate animal, further comprising the step of identifying a vertebrate animal in need of treatment of the infectious diesease.
  • administering comprises mixing an effective amount of a disclosed APB A therapeutic peptide with the food of the vertebrate animal.
  • administering comprises administering enterally an effective amount of the disclosed APBA therapeutic peptide with the food of the vertebrate animal.
  • administering comprises administering an oral bolus of an effective amount of the disclosed APBA therapeutic peptide with the food of the vertebrate animal.
  • administering to a vertebrate animal comprises intravenous administration or parenteral administration to the vertebrate animal.
  • the infectious disease treated in the vertebrate animal is selected from dental infection, dermatitis, diarrhea, ear infection, gastritis, gastroenteritis, genitourinary infection, intestinal infection, lung infection, ocular infection, oral infection, otitis, osteo-articular infection, pharyngitis, papules, pneumonia conjunctivitis, pruritius, pustules, pyoderma, pyothorax, respiratory infection, salmonellosis, septicemia, skin infection, soft tissue infection, ulcer, urinary tract infection, and wound infection.
  • the disclosure relates to a method for the treatment of an infectious disease in a vertebrate animal, further comprising administering to the vertebrate animal a therapeutically effective amount of second active agent.
  • the second active agent is an antibacterial agent.
  • the antibacterial agent is pencillin, a cephalosporin, a sulfonamide, a tetracycline, a lincosamide, an aminoglycoside, or a fluoroquinolone, or combinations thereof.
  • the antibacterial agent comprises a compound selected from amoxicillin, ampicillin, azithromycin, cefovecin, cephalexin, chloramphenicol, ciprofloxacin, clavulanic acid, cloxacillin, clindamycin, doxycycline, enrofloxacin, erythromycin, gentamicin, ibafloxacin, kanamycin, lincomycin, marbofloxacin, metronidazole, minocycline, neomycin, novobiocin, ofloxacin, orbifloxacin, oxytetracycline, penicillin G, rifampin, sulfadimethoxine, sulfadiazine, tetracycline, tiamulin, ticarcillin, trimethoprim, and tylosin, or combinations thereof.
  • the disclosed APBA therapeutic peptides are further useful in a method for the prevention, treatment, control, amelioration, or reduction of risk of the bacterial infections noted herein.
  • the disclosed APBA therapeutic peptides are further useful in a method for the prevention, treatment, control, amelioration, or reduction of risk of the aforementioned bacterial infections in combination with other agents.
  • the disclosed APBA therapeutic peptides can be used in combination with one or more other drugs in the treatment, prevention, control, amelioration, or reduction of risk of bacterial infections for which disclosed APBA therapeutic peptides or the other drugs can have utility, where the combination of the drugs together are safer or more effective than either drug alone.
  • Such other drug(s) can be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure.
  • a pharmaceutical composition in unit dosage form containing such other drugs and a disclosed compound is preferred.
  • the combination therapy can also include therapies in which a disclosed compound and one or more other drugs are administered on different overlapping schedules. It is also contemplated that when used in combination with one or more other active ingredients, the disclosed APBA therapeutic peptides and the other active ingredients can be used in lower doses than when each is used singly.
  • compositions include those that contain one or more other active ingredients, in addition to a compound of the present disclosure.
  • the above combinations include combinations of a disclosed compound not only with one other active compound, but also with two or more other active compounds.
  • disclosed APBA therapeutic peptides can be used in combination with other drugs that are used in the prevention, treatment, control, amelioration, or reduction of risk of the bacterial infections for which disclosed APBA therapeutic peptides are useful.
  • Such other drugs can be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present disclosure.
  • a pharmaceutical composition containing such other drugs in addition to a disclosed compound is preferred.
  • the pharmaceutical compositions include those that also contain one or more other active ingredients, in addition to a compound of the present disclosure.
  • the weight ratio of a disclosed compound to the second active ingredient can be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the present disclosure is combined with another agent, the weight ratio of a disclosed compound to the other agent will generally range from about 1000: 1 to about 1 : 1000, preferably about 200: 1 to about 1 :200. Combinations of a compound of the present disclosure and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
  • a disclosed compound and other active agents can be administered separately or in conjunction.
  • the administration of one element can be prior to, concurrent to, or subsequent to the administration of other agent(s).
  • the disclosed APBA therapeutic peptides can be used alone or in combination with other agents which are known to be beneficial in the subject indications or other drugs that affect receptors or enzymes that either increase the efficacy, safety, convenience, or reduce unwanted side effects or toxicity of the other agents.
  • the disclosed APBA therapeutic peptide and the other agent can be coadministered, either in concomitant therapy or in a fixed combination.
  • the compound can be employed in combination with antibacterial or antimicrobial agents, and combinations thereof, and the like, or the subject compound can be administered in conjunction with the use of physical methods such as with debridement of a wound or infected tissue.
  • an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses.
  • the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day.
  • a suitable dosage level can be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day.
  • compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15,20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • the compounds can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosage regimen can be adjusted to provide the optimal therapeutic response.
  • the disclosure relates to methods for treating a bacterial infection in at least one cell, comprising the step of contacting the at least one cell with at least one compound of the disclosure, in an amount effective to alter the response in the at least one cell.
  • the cell is mammalian, for example human.
  • the cell has been isolated from a subject prior to the contacting step.
  • contacting is via administration to a subject.
  • Infectious diseases treatable by the presently disclosed APBA therapeutic peptides can be caused by a variety of bacteria and protozoa.
  • the infection is a bacterial infection.
  • Exemplary microbial infections that can be treated by the method of the presently disclosed APBA therapeutic peptides include, but are not limited to, infections caused by Staphylococcus aureaus, Enterococcus faecalis, Bacillus anthracis , a Streptococcus species (e.g., Streptococcus pyogenes and Streptococcus pneumoniae ), Escherichia coli, Pseudomonas aeruginosa, Burkholderia cepacia , a Proteus species (e.g., Proteus mirabilis and Proteus vulgaris ), Klebsiella pneumoniae , Acinetobacter baumannii , Strenotrophomonas maltophillia ,
  • M. tuberculosis M. avium intracellular e
  • M. abscessus M. kansasii
  • M. fortuitum M. chelonae
  • M. leprae cases of human tuberculosis are predominantly caused by mycobacterial species comprising M. tuberculosis, M. bovis , or M. africanum.
  • Infection is typically initiated by the inhalation of infectious particles, which are able to reach the terminal pathways in the lungs.
  • the bacilli Following engulfment by alveolar macrophages, the bacilli are able to replicate freely, with eventual destruction of the phagocytic cells. A cascade effect ensues wherein destruction of the phagocytic cells causes additional macrophages and lymphocytes to migrate to the site of infection, where they too are ultimately eliminated.
  • Mycobacteria can be classified into several major groups for purpose of diagnosis and treatment: M. tuberculosis complex (MTBC), which can cause tuberculosis (M tuberculosis, M. bovis, M. africanum , and M. microti ); M. leprae , which causes Hansen's disease or leprosy; and Nontuberculous mycobacteria (NTM) are all the other mycobacteria, which can cause pulmonary disease resembling tuberculosis, lymphadenitis, skin disease, or disseminated disease.
  • MTBC members are causative agents of human and animal tuberculosis. Species in this complex include: M.
  • tuberculosis the major cause of human tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti , andM pinnipedii.
  • the present disclosure provides methods of treating a mycobacterial infections, including those caused by mycobacteria such asM tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium paratuberculosis, M. avium silvaticum, M. avium“homninissuis”, M. colombiense, M. asiaticum, M. gordonae, M. gastri, M. kansasii, M. hiberniae, M. nonchromogenicum, M. terrae, M.
  • mycobacteria such asM tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canetti, M. caprae, M. microti, M. pinnipedii, M. avium, M. avium parat
  • M. wolinskyi M. thermoresistibile, M. gadium, M. komossense, M. obuense, M. sphagni, M. agri, M. aichiense, M. alvei, M. arupense, M. brumae, M. canariasense, M. chubuense, M. conceptionense, M. duvalii, M. elephantis, M. gilvum, M. hassiacum, M. holsaticum, M. immunogenum, M. massiliense, M. moriokaense, M. psychrotolerans, M. pyrenivorans, M. vanbaalenii, M.
  • the present disclosure provides methods of treating an infectious disease such as a mycobacterial infection.
  • the mycobacterial infection can be associated with a Mycobacterium tuberculosis infection.
  • the Mycobacterium tuberculosis infection is associated with infection by an MDR strain of Mycobacterium tuberculosis.
  • the Mycobacterium tuberculosis infection is associated with infection by an XDR strain of Mycobacterium tuberculosis.
  • the present disclosure provides methods of treating an infectious disease such as a Gram positive bacterial infection.
  • the Gram positive bacteria is selected from Bacillus sp. Clostridium sp., Corynebacterium sp, Enterococcus sp., Mycoplasma sp., Staphylococcus sp., and Streptococcus sp.
  • the Gram positive bacteria is vancomycin resistant Enterococcus sp. (VRE).
  • the Gram positive bacteria is methicillin resistant Staphylococcus sp. (MRS).
  • the Gram positive bacteria is selected from Bacillus anthracis , Bacillus cereus , Bacillus subtilis , Clostridium difficile , Clostridium tetani , Clostridium botulinum , Clostridium perfringens , Corynebacterium diphtheria , Enterococcus faecalis , Enterococcus faecium , Listeria monocytogenes , Listeria ivanovii , Micrococcus luteus , Mycoplasma genitalium , Mycoplasma pneumoniae , Staphylococcus aureus , Staphylococcus epidermidis , Staphylococcus saprophyticus , Staphylococcus hyicus , Staphylococcus intermedins, Streptococcus pneumoniae , and Streptococcus pyogenes.
  • the Gram positive bacteria is selected from Bacillus anthracis , Bacillus subtilis , Enterococcus faecalis , Staphylococcus aureus , Streptococcus pneumoniae , and Streptococcus pyogenes.
  • the Gram positive bacteria is selected from vancomycin resistant Enterococcus faecalis , vancomycin resistant methicillin resi stant Enterococcus faecium , Staphylococcus aureus (MRS A), methicillin resistant Staphylococcus epidermidis (MRSE), macrolide resistant Streptococcus pneumoniae (Mac-R SPN) and penicillin resistant Streptococcus pneumonia (PRSP).
  • vancomycin resistant Enterococcus faecalis vancomycin resistant methicillin resi stant Enterococcus faecium
  • Staphylococcus aureus MRS A
  • methicillin resistant Staphylococcus epidermidis MRSE
  • Mac-R SPN macrolide resistant Streptococcus pneumoniae
  • PRSP penicillin resistant Streptococcus pneumonia
  • the present disclosure provides methods of treating an infectious disease such as a Gram negative bacterial infection.
  • the Gram negative bacteria is selected from ri cinetohacter sp., Aeromonas sp., Burkholderia sp., Bordatella sp., Citrobacter sp., Chlamydia sp., Enterobacter sp., Escherichia sp., Francisella sp., Haemophilus sp., Klebsiella sp., Legionella sp., Moraxella sp., Neisseria sp., Proteus sp., Pseudomonas sp., Rickettsia sp., Salmonella sp., Shigella sp., Stenotrophomonas sp., Vibrio sp., and Yersinia sp.
  • the Gram negative bacteria is selected from Acinetobacter baumannii , Aeromonas hydrophA a, Bordetella pertussis , Bordetella parapertussis , Bordetella bronchiseptica , Burkholderia cepacia , Citrobacter freundii , Chlamydia pneumoniae , Chlamydia trachomatis , Chlamydia psittaci , Enterobacter aerogenes, Enterobacter cloacae , Enterobacter sakazaku, Escherichia colt, Francisella tularensis , Haemophilus influenzae , Haemophilus aegypticus , Haemophilus ducreyi , Klebsiella edwardsii , Klebsiella pneumoniae , Legionella pneumophilia , Moraxella catarrhalis , Neisseria mening
  • the Gram negative bacteria is a multi-drug resistant Gram negative bacteria strain (MDR-GNB).
  • MDR-GNB multi-drug resistant Gram negative bacteria strain
  • the multi-drug resistant Gram negative bacteria strain (MDR-GNB) is resistant to at least one anti-microbial agent selected from amikacin, tobramycin, cefepime, ceftazidime, imipenem, meropenem, piperacillin-tazobactam, ciprofloxacin, levofloxacin, tigecy cline, and polymyxin B.
  • the multi-drug resistant Gram negative bacteria strain is selected from Acinetobacter sp., Enterobacter sp., Klebsiella sp., and Pseuodomonas sp.
  • the multi-drug resistant Gram negative bacteria strain is selected from Acinetobacter baumannii , Enterobacter aerogenes, Klebsiella pneumoniae , and Pseudomonas aeruginosa.
  • the multi-drug resistant Gram negative bacteria strain is Enterobacter sp.
  • the present disclosure provides methods of treating an infectious disease selected from atypical pneumonia, bacterial meningitis, bronchitis, cholera, dental infection, dermatitis, diarrhea, diphtheria, dysentery, ear infection, endocarditis, gastritis, gastroenteritis, genital infection, genitourinary infection, infection associated with an indwelling device, intestinal infection, leprosy, listeriosis, lung infection, nocosomial infection, ocular infection, oral infection, otitis, osteo-articular infection, osteomyelitis, pharyngitis, papules, pharyngitis, pneumonia, pneumonia conjunctivitis, pruritius, pustules, pyoderma, pyothorax, respiratory infection, salmonellosis, septicemia, sexually transmitted disease, sinusitis, skin infection, skin and soft tissue infection ("SSTI”), soft tissue infection, tetanus, tuberculo
  • SSTI soft tissue infection
  • the infectious disease is selected from endocardititis, osteomyelitis, skin and soft tissue infection ("SSTI"), and infection associated with an indwelling device.
  • the infectious disease is endocardititis.
  • the infectious disease is osteomyelitis.
  • the infectious disease is an SSTI.
  • the SSTI is a complicated SSTI (cSSTI).
  • the infectious disease is associated with an indwelling device.
  • the present disclosure provides methods of treating an infectious disease such in a human subject comprising administering a disclosed compound or a disclosed pharmaceutical composition, and further comprising administering to the human subject a therapeutically effective amount of a second active agent.
  • the second active agent comprises at least one antibacterial agent.
  • the antibacterial agent comprises a compound selected from amoxicillin, ampicillin, azithromycin, aztreonam, azlocillin, bacitracin, carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cefdinir, cefditorin, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, chloramphenicol, cilastin, ciprofloxacin, clarithromycin, clavulanic acid, clinafloxacin, clindamycin, clofazimine, cloxacillin, colistin, cycloserin, dalbavancin, dalfopristin
  • the present disclosure provides methods of treating an infectious disease such in a human subject comprising administering a disclosed compound or a disclosed pharmaceutical composition, and further comprising administering to the human subject a therapeutically effective amount of an anti-tuberculosis agent.
  • the anti tuberculosis agent is selected from amikacin, amoxicillin-clavulanic acid, bedaquiline, capreomycin, ciprofloxacin, clarithromycin, clofazimine, cycloserine, ethambutol, ethionamide, gatifloxacin, imipenem, isoniazid, kanamycin, levofloxacin, meropenem, moxifloxacin, ofloxacin, OPC-7683, para-aminosalicylic acid, pretomanid, pyrazinamide, rifampin, rifapentine, rifabutin, SQ109, streptomycin, sudoterb, terizidone, thiacetazone, viomycin, and combinations thereof.
  • the anti-tuberculosis agent is an aminoglycoside antibiotic, such as kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, paromomycin and streptomycin.
  • an aminoglycoside antibiotic such as kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycin B, neomycin C, paromomycin and streptomycin.
  • the anti-tuberculosis agent is a fluroquinolone, such as moxifloxacin, levofloxacin, sparfloxacin, nalidixic acid, ciprofloxacin, cinoxacin, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, perfloxacin, rufloxacin, balofloxacin, grepafloxacin, pazufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatlifloxacin, sitafloxacin, prulifloxacin, delafloxacin, JNJ-Q2, nemofloxacin, danofloxacin, difloxacin, enrofloxacin,
  • the present disclosure provides methods of treating an infectious disease such in a human subject comprising administering a disclosed compound or a disclosed pharmaceutical composition, and further comprising administering to the human subject a therapeutically effective amount of an immunomodulatory agent.
  • the immunomodulatory agent is a cytokine, an interleukin, a chemokine, or combinations thereof.
  • the immunomodulatory agent is selected from IL-2, IL-7 and IL-12, IFN-a, IFN-b, IFN-e, IFN-k, IFN-CO, IFN-g, IFN-g lb, CCL3, CCL26, CXCL7, and combinations thereof.
  • the administering is co-administering of the disclosed compound and the antibacterial agent.
  • the co-administration is administration in a substantially simultaneous manner of the disclosed compound and the antibacterial agent.
  • the co-administration is administration in a substantially sequential manner of the disclosed compound and the antibacterial agent.
  • the administration in a substantially simultaneous manner comprises a single dose form containing a fixed ratio of the compound and the antibacterial agent.
  • the single dose form is a capsule or a tablet.
  • the single dose form is an ampule for a single intravenous administration.
  • the disclosed APBA therapeutic peptides can have a mechanism of antimicrobial action and/or may bind to and/or inhibit one or more bacterial target molecules or macromolecular complexes containing a bacterial target molecule.
  • Mechanisms of action may include inhibiting or interfering with a biological or biochemical pathway of the bacterium.
  • Exemplary pathways include, but are not limited to, protein synthesis, cell wall synthesis, DNA replication, transcription, and cell division. It will be appreciated that biological and biochemical pathways are not mutually exclusive and that some biological or biochemical pathways may be considered to be subsets or sub-pathways of other biological or biochemical pathways.
  • Mechanisms of action include, but are not limited to, inhibiting protein synthesis (e.g., by binding ribosomal RNA or proteins, blocking tRNA binding to the ribosome-mRNA complex, inhibiting peptidyl transferase), inhibiting or interfering with synthesis of a cell wall component (e.g., inhibition of peptidoglycan synthesis, disruption of peptidoglycan cross-linkage, disruption of movement of peptidoglycan precursors, disruption of mycolic acid or arabinoglycan synthesis), cell membrane disruption, inhibiting or interfering with nucleic acid synthesis of processing, acting as“antimetabolites” and either inhibiting an essential bacterial enzyme or competing with a substrate of an essential bacterial enzyme, inhibiting or interfering with cell division.
  • protein synthesis e.g., by binding ribosomal RNA or proteins, blocking tRNA binding to the ribosome-mRNA complex, inhibiting peptidyl transferase
  • a cell wall component e.
  • Molecules, or macromolecular complexes containing them, that may be targets for antibiotics include, but are not limited to, peptidoglycans, penicillin binding proteins, lipopolysaccharides, ribosomes or ribosomal subunits or RNA or protein components thereof (23 S rRNA, 16S rRNA, proteins of the 3 OS or 50S subunit), DNA-dependent DNA polymerase, DNA- dependent RNA polymerase, microbial type I topoisom erase, microbial type II topoisomerase (e.g., topoisomerase IV or gyrase), enzymes involved in cell division such as FtsZ, etc.
  • peptidoglycans penicillin binding proteins, lipopolysaccharides, ribosomes or ribosomal subunits or RNA or protein components thereof (23 S rRNA, 16S rRNA, proteins of the 3 OS or 50S subunit)
  • DNA-dependent DNA polymerase DNA-dependent RNA poly
  • the disclosed APBA therapeutic peptides inhibit bacterial protein synthesis.
  • the bacterial species may be of any one or more types, e.g., gram-negative bacteria, gram-positive bacteria, atypical bacteria, and/or acid fast bacteria.
  • Suitable organisms can include, but are not limited to members of the following genuses: Actinomyces, Staphylococcus, Streptococcus, Enterococcus, Erysipelothrix, Neisseria, Branhamella, Listeria, Bacillus, Corynbacterium, Erysipelothrix, Gardnerella, Mycobacterium, Nocardia, Enter obacteriaceae, Escherichia, Salmonella, Shigella, Yersinia, Enterobacter, Klebsiella, Citrobacter, Serratia, Providencia, Proteus, Morganella, Edwardsiella, Erwinia, Vibrio, Aeromonas, Helicobacter, Campylobacter, Eikenella, Pasteurella, Pseudomonas, Burkholderia, Stenotrophomonas, Acinetobacter, Ralstonia, Alcaligenes, Moraxella, Mycoplasma, Legionella, Francisella, Bruce
  • the bacteria are species that are causative agents of disease in humans and/or animals. Examples include, but are not limited to, Acinetobacter baumannii, Aeromonas hydrophila, Bacillus anthracis, Bacillus anthracis sterne, Bacillus subtilis, Burkholderia cepacia, Escherichia coli, Enterobacter cloacae, Enterococcus faecalis, Francisella tularensis, Campylobacter jejuni, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella oxytoca, Legionella pneumophila, Pasteurella multocida, Proteus mirabilis, Proteus vulgaris, Mycobacterium tuberculosis, Morganella morganii, Helicobacter pylori, Neisseria meningitides, Neisseria gonorrhoeae, Chlamydia trachomatis,
  • the present disclosure pertains to uses of a disclosed APBA therapeutic peptide, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament with a pharmaceutically acceptable carrier or diluent for the treatment of a disorder associated with a microbial infection in a mammal, e.g., a human.
  • the present disclosure pertains to methods for the manufacture of a medicament to treat an infection associated with an antibiotic resistant microbe comprising combining at least one disclosed compound, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament with a pharmaceutically acceptable carrier or diluent.
  • the disclosure relates to a medicament comprising one or more disclosed APBA therapeutic peptides; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.
  • the disclosure relates methods for the manufacture of a medicament for the treatment of a disorder associated with a microbial infection in a mammal (e.g., treatment of one or more bacterial infections) in mammals (e.g., humans) comprising combining one or more disclosed APBA therapeutic peptides, or a pharmaceutically acceptable salt, solvate, hydrate, or polymorph thereof, and at least one additional therapeutic agent with a pharmaceutically acceptable carrier. Kits
  • kits comprising at least one disclosed compound, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, and one or more of: (a) at least one agent known to treat a disorder associated with a microbial infection; or (b) instructions for treating a disorder associated with a microbial infection.
  • kits whereby two or more components, which may be active or inactive ingredients, carriers, diluents, and the like, are provided with instructions for preparation of the actual dosage form by the patient or person administering the drug to the patient.
  • kits may be provided with all necessary materials and ingredients contained therein, or they may contain instructions for using or making materials or components that must be obtained independently by the patient or person administering the drug to the patient.
  • kits can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, a kit can contain instructions for preparation and administration of the compositions.
  • the kit can be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple patients (“bulk packaging”).
  • the kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.
  • kits can be packaged in a daily dosing regimen (e.g., packaged on cards, packaged with dosing cards, packaged on blisters or blow-molded plastics, etc.).
  • a daily dosing regimen e.g., packaged on cards, packaged with dosing cards, packaged on blisters or blow-molded plastics, etc.
  • Such packaging promotes products and increases patient compliance with drug regimens.
  • Such packaging can also reduce patient confusion.
  • the present invention also features such kits further containing instructions for use.
  • the present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits can also comprise compounds and/or products co- packaged, co-formulated, and/or co-delivered with other components.
  • a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.
  • kits can be used in connection with the disclosed methods of making, the disclosed methods of using or treating, and/or the disclosed compositions.
  • the disclosed APBA therapeutic peptides and pharmaceutical compositions have activity as anti-microbial therapeutic agents.
  • the disclosed APBA therapeutic peptides are also useful as research tools.
  • one aspect of the present disclosure relates to a method of using a disclosed APBA therapeutic peptide as a research tool, the method comprising conducting a biological assay using a disclosed APBA therapeutic peptide in an anti-microbial assay and determining microbial growth.
  • disclosed APBA therapeutic peptides can also be used to evaluate new chemical compounds.
  • Another aspect of the invention relates to a method of evaluating a test compound in a biological assay, comprising: (a) conducting a biological assay with a test compound to provide a first assay value; (b) conducting the biological assay with a disclosed APBA therapeutic peptide to provide a second assay value; wherein step (a) is conducted either before, after or concurrently with step (b); and (c) comparing the first assay value from step
  • Still another aspect of the invention relates to a method of studying a biological system, e.g., a model animal for a clinical condition, the method comprising: (a) contacting the biological system with a disclosed APBA therapeutic peptide; and
  • the Ph.D.TM-C7C Phage Display Peptide Library Kit and the E. coli K12 ER2738 strain were purchased from New England Biolabs. All ER2738 cultures were grown in the presence of 20 pg/mL tetracycline. Chemical reagents for small molecule, library synthesis and confirmation and peptide synthesis were purchased from various vendors and used as received. The fluorescent gel was imaged on a BioRad ChemiDoc MP Imaging System. The S. aureus (ATCC 6538) and MRS A (ATCC 43300) were purchased from Microbiologies as a lyophilized pellet. The wild-type A.
  • baumannii (AB5075) is a virulent and multidrug resistant clinical isolate that has been established as a pathogenic model strain and the LOS deficient baumannii (5075 LOS-) was established from AB5075 through selection for colistin resistance.
  • NMR data of the small molecule was collected on a VNMRS 500 MHz NMR spectrometer.
  • Peptide synthesis was performed on a Tribute peptide synthesizer from Protein Technologies and purified via reverse- phase high performance liquid chromatography (RP-HPLC) on a Waters Prep LC with a Jupiter C18 Column (Phenomenex). Mass spectrometry data were generated using an Agilent 6230 LC TOF mass spectrometer.
  • Fluorescence images were captured on a Zeiss Axio Observer Al inverted microscope. Flow cytometry analysis was performed on a BD FACSAria cell sorter. Photoinactivation was performed with a X-Cite 120Q (l20-Watt lamp) excitation light source accompanied with the Zeiss microscope.
  • Jurkat T lymphocytes were a gift from the Johnson lab at Boston College, HEK293T cells were a gift from the Weerapana lab at Boston College, and mammalian cell cytotoxicity was evaluated on a SpectraMax M5 plate reader along with fluorescence anisotropy.
  • the Ph.D.TM-C7C Phage Display Peptide Library (5 pL, ⁇ lxl0 13 pfu/mL) was subjected to reduction in the presence of iTCEP (25 pL), in a total volume of 200 pL in TBS (pH 8.5) for 48 hours at 4°C.
  • APBA-IA (2 mM, 2 pL from a 200 mM DMSO stock) was added to the reduced phage and allowed to conjugate for 2 hours at room temperature.
  • the labeled phage was removed from iTCEP and precipitated to remove excess labeling reagent with 1/6 volume 20% (w/v) PEG- 8000, 2.5 M NaCl for 5 hours at 4°C.
  • Precipitated phage was re-dissolved in PBS (pH 7.4, 100 pL) and the phage titer was calculated according to the Ml 3 Titer Protocol provided by New England Biolabs.
  • APBA-IA and Biotin-IA labeled library phage ( ⁇ lxl0 10 pfu/mL) were subjected to labeling with Scz-FITC (2 mM), synthesized previously, for 1 hour followed by precipitation. Phage samples were heat denatured at 95 °C for 5 minutes. Samples were subjected to 15% SDS-PAGE for 50 minutes, allowing the lower molecular weight PVIII protein to run off the gel, and imaged.
  • S. aureus was grown in LB medium to an OD 6 oo ⁇ l .O ( ⁇ lxl0 9 cfu/mL).
  • the cells (1 mL) were washed with chilled PBS containing 0.05% Tween (PBST) twice and resuspended in PBS (pH 7.4) with 10 mg/mL BSA present.
  • the APBA-labeled phage library ( ⁇ lxl0 10 pfu) was added to the cell suspension and allowed to incubate on ice for 1 hour. The cells were washed three times with PBST and three times with PBS to remove unbound phage.
  • Cell-bound phage were incubated with 200 pL elution buffer (Glycine-HCl, pH 2.2, 1 mg/mL) for 15 minutes followed by centrifugation of the cells. The supernatant was removed and neutralized with 150 pL Tris-HCl (pH 9.1). All Eppendorf tubes utilized in the panning procedure were blocked with 10 mg/mL BSA before use. Centrifugation of cells was performed at 5,000 ref for 5 minutes. The eluted bound phage solution was added to early-log phase ER2738 and amplified for 4.5 hours followed by precipitation to isolate the amplified phage. The amplified phage were labeled with APBA-IA and subjected to the next round of panning.
  • elution buffer Glycine-HCl, pH 2.2, 1 mg/mL
  • the phage titer was calculated before and after each round of panning to determine the input and output population. Individual phage colonies from each round of panning were amplified in ER2738. Phage DNA was isolated using a Qiagen miniprep kit and sent for sequencing analysis by Eton Bioscience, Inc. The screens against S. aureus with the unmodified C7C library and the IA-alkylated library (C7C-IA) were performed following the same protocol.
  • the C7C-IA library was prepared using the same protocol described for the APBA- dimer library preparation.
  • the screen against A. baumannii (LOS-) was performed following the same protocol; however, a negative screen was introduced against A. baumannii (LOS+) starting in the second round. In the negative screen, the phage library was incubated with A. baumannii (LOS+) for 1 hour, the supernatant was removed and subsequently subjected to the positive screen against A. baumannii (LOS-).
  • Each bacterial strain was grown to an OD 6 oo ⁇ 0.5, washed and diluted with PBS (pH 7.4).
  • the cells ( ⁇ lxl0 7 cfu/mL) were incubated with various concentrations of FAM-labeled peptide with or without BSA in PBS. After incubation for 1 hour, samples were subjected to cytometric analysis. Data obtained was analyzed via BD FACSDiva software and median fluorescent values were computed from the generated histograms. All flow cytometry experiments were repeated and generated consistent results (see Figure 14).
  • Each bacterial strain was grown to an OD 6 oo ⁇ l .O, washed and diluted with PBS (pH 7.4).
  • the cells ( ⁇ lxl 0 9 cfu/mL) were incubated with various concentrations of TAMRA-labeled peptide with or without BSA in PBS for 1 hour.
  • the same microscopy procedure described was implemented using filter set 20 HE (excitation: BP 546/12, emission: BP 607/80) suitable for detection of TAMRA fluorescence and images were processed consistently using ImageJ software.
  • Each bacterial strain was grown to an OD 6 oo ⁇ 0.7, washed and diluted with PBS (pH 7.4).
  • the cells ( ⁇ lxl0 8 cfu/mL) were incubated with eosin-conjugated peptides and various controls for 15 minutes.
  • Half of the bacterial suspension was removed and placed in a 96-well plate (Coming 3595). The well was subjected to photoirradiation on the Zeiss microscope using the 20X objective and fluorescein filter to emit blue light for 15 minutes.
  • Cells were diluted in LB media, spread on LB agar plates and incubated overnight at 37°C. The A.
  • baumannii (LOS-) strain was spread on LB agar (+ 10 pg/mL polymyxin b) and incubated for 24 hours. The amount of cell killing was calculated by comparing the amount of colonies of treated bacteria to an untreated PBS control. All experiments were repeated and the average cell killing of two trials was plotted.
  • the solution was acidified to pH 3 by 1 N HC1 and the product was extracted with DCM (3 x 100 mL). The combined organic layer was washed with brine (100 mL) and dried over sodium sulfate. DCM was removed and the residue was treated with TFA/H2O for 2 h. After solvent removal, the crude material was re-dissolved in 10 mL Acetonitrile/H20 (2:3) solution and purified via RP-HPLC. The product is a white solid after lyophilization (85 mg, 35% yield over three steps).
  • Streptavidin agarose resin 25 pL/sample was washed with PBS (pH 7.4) and blocked with 10 mg/mL BSA via incubation for 1 hour.
  • APBA-IA labeled library was subjected to subsequent labeling with Biotin-IA (2 mM) for 2 hours followed by precipitation.
  • Biotin-IA labeled and APBA-IA/Biotin-IA labeled phage 200 pL, -lxlO 10 pfu/mL were subjected to the streptavidin resin for 1 hour. Non-reduced and reduced phage, without small molecule labeling, were also analyzed.
  • Unbound phage was removed from resin and the phage titer was calculated. The titer was compared to that of phage not subjected to streptavidin to generate a percent capture. The average percent capture and standard deviation of three trials was plotted. Wild-type phage, with no library insert, was subjected to the same analysis for comparison.
  • Fluorescein labeled anti-Ml3 major coat protein antibody (1 pg, Santa Cruz Biotechnology) was added to the bacterial suspension, incubated for 30 minutes and directly subjected to fluorescence microscopy analysis. Antibody binding to S. aureus , with no phage present, was also analyzed to assess any background fluorescence.
  • White light and fluorescent images were obtained on the Zeiss microscope equipped with filter set 44 (excitation: BP 475/40, emission: BP 530/50) suitable for detection of fluorescein fluorescence. Images were captured using the 100X oil immersion objective with a 500 ms exposure time. All images were processed consistently using ImageJ software.
  • 5(6)-FAM, 5(6)-TAMRA and 5(6)-carboxyeosin were conjugated to the peptide on resin by first removing the alloc protecting group with tetrakis(triphenylphosphine)palladium(0) and phenylsilane in DCM followed by subsequent HBTU-mediated amide bond coupling in 0.4 M NMM/DMF.
  • the peptides were cleaved off resin and globally deprotected with reagent B (88% TFA, 5% H20, 2% triisopropylsilane, 5% phenol). Crude peptides were obtained via ether precipitation and purified by RP-HPLC.
  • each peptide hit was treated with 3 equivalents of APBA-IA in the presence of TCEP (2 eq) in 2 M NMM/DMF for 3 hours and purified via RP-HPLC. All peptides were characterized with LC-MS to confirm their identities and excellent purities (>95%).
  • Jurkat cells were cultured in RPMI 1640 (containing 10% FBS, 2 mM glutamine, 1% Penicillin/Streptomycin) and maintained at 5xl0 5 cells/mL. Cells were diluted in RPMI and distributed to a 96-well plate (Coming 3595) at 50,000 cells/well (200 pL/well). 2 pL of a 100X DMSO solution of KAM5-Eosin (200 pM) and KAM8-Eosin (200 pM) was added to each well and incubated for 24 hours. A positive control for viability of DMSO treated cells along with a positive control for cytotoxicity of camptothecin at 50 pM (5 mM DMSO stock) were included.
  • the cells were incubated with the compound of interest for 15 minutes, each well was irradiated for 15 minutes (see photoinactivation protocol -paragraph 0053) and the plate was returned to the incubator for the remainder of the 24 hour incubation. Cells were centrifuged for 5 minutes (180 ref) and the supernatant culture medium was carefully removed. 100 pL of 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg/mL in RPMI) was added to each well and incubated for 4 hours followed by the addition of 10% SDS in 0.01 M HC1 (100 pL) and incubation overnight.
  • MTT 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • HEK293T cells were grown in complete DMEM (containing 10% FBS, 2 mM glutamine and 1% Penicillin/Streptomycin) to about 80% confluency in a 150 mm dish. Cells were removed from the plate with 0.25% trypsin protease solution (containing EDTA) at 37°C for 5 minutes and pelleted (3,500 rpm, 5 minutes). Cells were diluted in DMEM, distributed to a 96-well plate (Corning 3595) at 30,000 cells/well (100 pL/well) and incubated for 24 hours to allow for cell adherence. 1 pL of 100X DMSO solutions were added to each well and incubations/photoirradiation were performed as described above.
  • complete DMEM containing 10% FBS, 2 mM glutamine and 1% Penicillin/Streptomycin
  • the present invention shows that incorporating an APBA warhead into cationic peptides can yield selective probes of gram-positive bacteria, which readily label a target bacterium in serum via a combination of reversible covalent and noncovalent interactions. It is envisioned that introducing such reversible covalent warheads into phage libraries could give a versatile platform to allow discovery of specific probes for diverse bacterial species and strains.
  • the phage library chosen for modification was the commercially available Ph.D.-C7C library, which displays disulfide-cyclized peptides with seven randomized residues that are fused to the pill minor coat protein of the Ml 3 phage.
  • the library was modified with APBA moieties via disulfide reduction and selective cysteine alkylation originally described by Derda and coworkers. Briefly, the disulfide bond of the C7C peptides was selectively reduced on phage with immobilized TCEP (iTCEP) for 48 hours at 4°C.
  • iTCEP immobilized TCEP
  • the reduced cysteines were then alkylated with an APBA derivative, namely APBA-IA, for 2 hours to yield the APBA-dimer library ( Figure 1B).
  • the details of APBA-IA synthesis can be found in Figures 6-8.
  • the extent of APBA-IA labeling was monitored by a pulse-chase assay in which biotin-iodoacetamide (Biotin-IA) treatment and streptavidin capture after APBA-IA labeling allowed quantification of phage that APBA-IA failed to label.
  • Biotin-IA biotin-iodoacetamide
  • streptavidin capture of the APBA-IA treated phage indicates complete labeling of cysteines by APBA-IA ( Figure 9).
  • Panning phage libraries directly against live bacterial cells presents an intriguing alterative to panning against target biomolecules.
  • earlier efforts along this front have only yielded low affinity (sub to low millimolar) peptide probes.
  • panning the APBA- dimer library against S. aureus has the potential to discover highly potent and selective peptide probes for this bacteria because it is known to overexpress lysine modified phosphoglycerol (Lys- PG) to afford resistance to host defense peptides.
  • Lys-PG synthesis is one of the critical features of S. aureus that makes it a prevalent pathogen.
  • the APB A-dimer library was screened against S. aureus cells in a suspension containing 10 mg/mL bovine serum albumin (BSA) as an internal competitor (Figure 2A).
  • BSA bovine serum albumin
  • Three rounds of affinity selection were initiated with an input population of 10 10 plaque forming units (pfu) in each round along with extensive washing steps to eliminate non-binders and strong albumin binders by centrifugation.
  • Acid treatment which is known to disrupt iminoboronate formation, was used to release bound phage from S. aureus.
  • the output population typically ranged from 10 3 to 10 5 pfu for S. aureus panning.
  • the recovered phage were amplified, labeled with APBA-IA and subjected to the next round of panning. After each round of panning, 10-20 colonies were randomly selected from the output population and subj ected to sequencing. Several peptide sequences were observed repeatedly in round 2 and round 3 even within this small set of colonies subjected for sequencing (Table 1). To determine which sequences merited further pursuit, a phage-based microscopy experiment was performed. The individual phage hits were modified with APBA-IA, incubated with S. aureus , and subsequently treated with a FITC-labeled anti-Ml3 antibody, which binds to the pVIII protein of the M13 phage. The phage- bound S.
  • Table 1 below shows sequences (SEQ ID NOS: 1-83, respectively, in order of columns) of the peptide hits for S. aureus binding (a) from round 2. (b) from round 3. (c) recurring sequences and frequency.
  • Table 2 shows synthesized peptide hits from S. aureus screening (SEQ ID NOS: 84- 88, respectively, in order of appearance).
  • Cm APBA-IA modified cysteine; *: fluorophore modified.
  • Table 3 shows mass-spec data of peptides prior to (a) and after (b) APBA-IA labeling (SEQ ID NOS: 89-106, respectively, in order of appearance). Dap s : FAM labeled; Dap : TAMRA labeled; Dap*: Eosin labeled. Cm: APBA-IA modified cysteine.
  • the bacterial binding potency displayed by KAM5 is orders of magnitude better than the peptides borne out of previous phage display efforts with natural peptide libraries, which only yielded sub to low mM binders.
  • the S. aureus binding potency of KAM5 is also much greater than that of Hlys-AB l, a rationally designed peptide that incorporates a single APB A motif ( Figure 15).
  • a negative control peptide KAM6 which was not selected from the screen and contains a random heptapeptide sequence, showed no bacterial staining under the same experimental conditions (Figure 2C).
  • the cyclic precursor of KAM5 (KAM5-Cyclic which has no APBA moieties) elicited little S. aureus staining as well, demonstrating the importance of the APBA warhead (Figure 2C).
  • KAM5 The cell staining ability of KAM5 was also evaluated via fluorescence microscopy with a TAMRA labeled peptide.
  • the microscopy studies yielded results consistent with those of flow cytometry: KAM5 at 2 mM concentration gave strong fluorescence staining of S. aureus cells and the addition of BSA up to 10 mg/mL did not inhibit the bacterial staining by KAM5. On the contrary, the BSA addition elicited stronger fluorescence staining of the bacteria, consistent with the flow cytometry results (Figure 2D).
  • the protein-enhanced bacterial binding by KAM5 is not BSA-specific as similar enhancement was observed with human serum albumin (HSA) as well ( Figures 16A-16B).
  • KAM5 was measured binding to these serum proteins via a fluorescence anisotropy experiment ( Figures 16A-16B). KAM5 did show binding to both BSA and HAS at high protein concentrations, which is perhaps not surprising given these proteins display a large number of surface lysine residues.
  • KAM5 binds the bacteria via covalent conjugation to Lys-PG, which is present on essentially all S. aureus strains, although its percentage may vary.
  • the bacterial selectivity of KAM5 toward different bacterial species was further analyzed.
  • Table 4 shows sequences (SEQ ID NOS 107-146, respectively, in order of columns) obtained from panning the unmodified C7C library against S. aureus (a) from round 2. (b) from round 3. (c) recurring sequences and frequency.
  • Table 5 shows sequences (SEQ ID NOS: 147-185, respectively, in order of columns) obtained from panning the C7C-IA library against S. aureus (a) from round 2. (b) from round 3. (c) recurring sequences and frequency.
  • Table 6 shows sequences (SEQ ID NOS: 186-189, respectively, in order of appearance) and mass-spec data of representative peptide hits from S. aureus screening with the C7C library (a) and C7C-IA library (b).
  • KAM14 and KAM15 are the two recurring sequences from round 3 of the C7C library.
  • the C7C-IA library only yielded one recurring sequence (KAM16) after round 3.
  • An additional peptide (KAM17) was randomly chosen from the round 3 output population for analysis.
  • Dap* FAM labeled Dap
  • Cm IA modified cysteine
  • a potent and selective S. aureus binder has the potential to serve as a directing element to develop targeted antibiotics.
  • the preferential binding of KAM5 towards S. aureus over serum proteins as well as other bacterial species makes it an excellent candidate for delivering a nonselective antibiotic to target cells.
  • KAM5 was conjugated to eosin, a phototoxin that upon photoirradiation triggers the production of reactive oxygen species (ROS), killing cells in close proximity (Figure 4A).
  • ROS reactive oxygen species
  • A. baumannii has emerged as a major healthcare-associated pathogen, which can cause severe infections in lungs and blood.
  • A. baumannii often presents resistance to multiple antibiotics, sometimes even to colistin (polymyxin E), one of the last-resort antibiotics for its treatment.
  • A. baumannii can acquire colistin resistance by modifying its lipooligosaccharide (LOS) with the addition of phosphoethanolamine or 4-aminoarabinose functionalities. Some strains even shut down LOS biosynthesis completely and replace the exterior leaflet of the outer membrane with lipoproteins.
  • LOS lipooligosaccharide
  • the APBA-dimer library on phage was screened against a LOS- mutant of A. baumannii (AB5075, a highly virulent isolate established as a model strain for A. baumannii infection). Three rounds of panning against the LOS-ri baumannii were executed following the same panning procedure described above for S. aureus except the addition of a negative screen against the wild-type (LOS+) A. baumannii in the second round. After each round of panning, 15 colonies were isolated from the output population and subjected to sequencing, in which convergence was detected starting in round 2 (Table 7). Four different peptide sequences (KAM7- 10) were observed repeatedly and synthesized via solid-phase peptide synthesis following the same procedure used for the S.
  • Table 7 shows sequences (SEQ ID NOS: 190-251, respectively, in order of columns) of the peptide hits obtained from screening the APBA-dimer library against A. baumannii (a) from round 2. (b) from round 3. (c) recurring sequences and frequency.
  • Table 8 shows synthesized peptide hits for A baumannii (LOS-) hits binding (SEQ ID NOS: 252-265, respectively, in order of appearance). Shown are sequences and mass-spec data prior to (a) and after (b) APB A-IA labeling.
  • Dap * FAM labeled
  • Dap* TAMRA labeled
  • Dap* Eosin labeled peptides.
  • Additional phage libraries can be developed for the following reasons: 1) given the vast variations between bacterial species and strains, having a collection of phage libraries can maximize the chance of success for identifying specific probes for new emerging strains of pathogens; 2) as shown in the present invention, screening of the APB A dimer library was able to give bacterial cell binders with low to sub mM potency. While these are on par with the Minimal Inhibitory Concentration (MIC) of many clinically used antibiotics, improving their potency can reduce the toxicity of the peptide-antibiotic conjugates for in vivo applications. For example, higher potency of peptide-colistin conjugates may avoid the well-known nephrotoxicity of the colistin itself; 3) peptide libraries of cyclic scaffolds are expected to yield improved biostability against protease degradation.
  • MIC Minimal Inhibitory Concentration
  • cyclic peptides are attractive as they often exhibit enhanced resistance toward protease cleavage.
  • the cyclic scaffold can preorganize the molecule for target binding as well.
  • a monocyclic peptide library can be readily constructed by treating the reduced C7C library with a bis-iodoacetamide (blA) derivative, which reacts with the reduced cysteine side chains to give a cyclic product ( Figure 23A).
  • the blA derivative (APBA-blA, Figure 23B) can then be synthesized to incorporate 2-APBA as a reversible covalent warhead.
  • a model C7C peptide can be used to determine the optimal concentration and conditions needed for phage modification.
  • the success of 2-APBA incorporation can be confirmed with semicarbazide ligation as was demonstrated in Figures 1B-1D for the APB A dimer library.
  • Phage displayed bicyclic peptide libraries that display reversible covalent warheads can also be developed.
  • the elegant work by Heinis et al. allowed the display of bicyclic peptides by crosslinking three cysteine residues that are strategically incorporated into the phage-displayed peptides. Currently, this remains the only method for presenting and evaluating multicyclic peptide libraries via phage display.
  • the Heinis system is less ideal for several reasons.
  • the engineered Ml 3 phage exhibit slow kinetics to infect E. coli for amplification, which is problematic as it gives growth advantage to the unmodified phage.
  • the simultaneous crosslinking of three cysteine residues makes it difficult to incorporate any additional binding motifs, such as the 2-APBA warhead, to better promote target binding.
  • the iminoboronate linkage although dynamic under physiologic conditions, can be rapidly and quantitatively reduced with NaCNBFb to afford permanent cyclization. Reduction of the iminoboronate linkage gives an o/V/ro-ami nomethyl -phenyl boronic acid moiety, which potentially forges an even stronger dative bond than that of iminoboronates. Supporting this notion, literature data show that ortho- boronic acid substituted benzyl amines can only be protonated at pH 2 or lower, while secondary amines typically display pKa values over 10. The strong B-N dative bond may render structural rigidity to the bicyclic peptides, which is an intrinsic advantage of this iminoboronate-based bicyclization strategy.
  • the iminoboronate-mediated bicyclization can be implemented on phage, which can yield a novel bicyclic peptide library. It is important to note that the iminoboronate-bicyclized peptides could still allow subsequent introduction of 2-APBA as a reversible covalent warhead to bind biological amines. Peptide bicyclization can be realized on phage by incorporating 2-APB A-lysine pairs at appropriate positions. Given reduction of iminoboronates is needed to afford the final permanently cyclized peptides, whether the Ml 3 phage could survive the NaCNBH 3 treatment was first tested. Titering results ( Figure 25) show that the phage library treated with NaCNBH 3 gives comparable colony count to the untreated control, indicating the Ml 3 phage is able to survive the NaCNBH3 treatment without significant damage.
  • an iodoacetamide derivative of AB3, AB3-IAA ( Figure 26) can be synthesized, which can alkylate cysteine on phage analogous to APBA-IA.
  • the side chain of the cysteine conjugate of AB3-IAA (named herewith as“CAB3”) can crosslink with a proximal lysine to give peptide cyclization.
  • CAB3 cysteine conjugate of AB3-IAA
  • the design of AB3-IAA incorporates an alkyne handle to enable further functionalization of the bicyclized peptides on phage.
  • Phage libraries can be created to display peptides with the sequence of ACXi . . .
  • XnCGKX n+i . . . Xn+mK SEQ ID NO: 270.
  • the randomized residues between the two cysteine residues can vary from 1 to 4 (n: 1-4).
  • the randomized residues between the two lysines can vary from 1 to 4 as well (m: 1-4).
  • An additional complication might result from the side chain flexibility of CAB3, which is longer and more flexible than that of AB3.
  • the increased side chain flexibility might allow it to crosslink with either one of the two lysines in the peptide sequence. Should that be the case, the central glycine in the CGK segment can be eliminated. Alternatively, the glycine residue could be replaced with a proline, which might effectively prevent cyclization within the (CAB3)PK segment.
  • the peptide sequences that are confirmed to undergo facile bicyclization can be introduced to the N-terminus of the pill protein using the Peptide Display Cloning System from New England Biolabs.
  • the NNC codon set can be used, which allows for the incorporation of 15 amino acids.
  • the choice of NNC codon is to exclude lysine from the randomized positions, which may cause complications to the regioselective bicyclization as expected for the chosen peptide sequence.
  • An eight-residue spacer (GGGSIDGR (SEQ ID NO: 266, Figure 26) can be further introduced between the displayed peptide and the pill protein.
  • This design can place the cysteine residues, which can later be converted to CAB3 residues, distant enough from the pill protein so that the 2-APBA moieties are not crosslink with native lysine residues of the phage (iminoboronate formation is reversible and distance dependent).
  • this spacer sequence incorporates a factor Xa cleavage site (IDGR (SEQ ID NO: 267)), which can allow the peptide to be cleaved off phage for mass-spec analysis.
  • IDGR factor Xa cleavage site
  • the phage library with the designed peptide sequences can be subjected to reduction and labeling with AB3-IAA.
  • the completeness of labeling can be confirmed with the established protocol used to characterize the APBA dimer library as shown in Figures 1B-1D. Then the AB3-IAA labeled phage can be subjected to NaCNBFb reduction to afford peptide bicyclization on phage. The bicyclic peptide library can then be tested for infectivity and amplification efficiency through titering. The peptide bicyclization on phage can be further confirmed via large scale preparation of a bicyclized phage, which can be treated with factor Xa and subjected to mass-spec analysis.
  • a pair of 2-APBA moieties can be installed through the azide-alkyne click chemistry.
  • the azido derivative of 2-APBA shown in Figure 26 can be made by derivatizing 2-acetyl-4-aminomethyl-phenol, an intermediate of APBA- blA synthesis as shown in Figure 23B. Conversion of the amine to azide followed by triflation and borylation can give the desired product for phage modification.
  • the success of click chemistry can be validated with mass-spec analysis of the factor Xa cleaved peptides.
  • the utility of the constructed phage libraries can be assessed by screening for potent bacterial binders in comparison to the APBA dimer library.
  • the screening can be performed using S. aureus and A. baumannii ( LOS- ) as the initial set.
  • the screening can be performed following the same protocol used for the APBA dimer library.
  • the peptide hits obtained from the cyclic peptide libraries can be synthesized by using similar protocols as developed for KAM5 and KAM8 synthesis. With fluorophore labeling, the peptide hits can be assessed for bacterial cell binding with florescence microscopy and flow cytometry. The potency of the cyclic peptide hits can be compared to those identified from the APBA dimer library.
  • the serum stability of the peptide hits from different phage libraries can be comparatively examined, and can be performed by using a standard protocol. Briefly, the fluorophore labeled peptides can be incubated with human blood serum and the percentage of intact peptides can be assessed by HPLC over time.
  • the cyclic peptide hits, particularly the bicyclic peptide hits, are anticipated to show longer half-life in human serum.
  • the general applicability of the phage display platform can be probed by examining a panel of bacterial species and strains.
  • a library screening can be performed against Streptococcus pneumoniae , which causes over 1.2 million drug resistant infections annually in the US.
  • a daptomycin insensitive and a daptomycin-sensitized strain can be screened against in parallel for comparison.
  • a recent publication indicates the significance of genes responsible for cell wall integrity in daptomycin sensitivity, although the chemical and structural basis of daptomycin resistance as well as its mode of action remains unclear.
  • the peptide hits identified can be tested for binding the S. pneumoniae strains as well as for binding S. aureus for comparison. The comparative study could reveal the bacterial binding potency and specificity of the peptide hits.
  • the library screening can be extended to the wild type A. baumannii (AB5075, LOS+), as well as several additional gram-negative pathogens including E. coli , K. pneumoniae , and P. aeruginosa. While not having a protein-coated surface, these gram-negative bacteria could be targeted by a peptide probe binding specific outer membrane proteins. This is possible given that POL7080, a targeted antibiotic for P. aeruginosa currently in clinical trials, was developed to specifically bind and inhibit the lipopolysaccharide transport protein LptD with nanomolar potency.
  • gram-negative bacteria In addition to the outer membrane proteins, gram-negative bacteria often display phosphoethanolamine modified LOS (or lipopolysaccharide (LPS), as shown in Figure 31, which elicits colistin resistance.
  • modified LOS can be efficiently targeted by iminoboronate-capable peptides, analogous to the S. aureus binders.
  • the phage library screening can be further extended to clinical strains of A. baumannii that are colistin-resistant and known to have LOS modifications. Given the success described in the present invention, potent and selective binders can be identified for these particular strains. The peptide hits identified from all the screens can be synthesized, fluorophore labeled, and then examined for binding the target and non-target strains. Comparative studies across this panel of bacteria could give a clear understanding on the scope and limitations of the phage display platform for targeting specific bacteria.
  • peptide hits An important aspect of characterizing the peptide hits is to elucidate the molecular targets of the identified peptide probes.
  • the peptide probes are designed to bind bacteria through formation of iminoboronates, which can be readily reduced to yield a permanent linkage. This unique property can allow facile identification of the molecular target(s) of the peptide probes.
  • an alkyne handle can be incorporated through an orthogonally protected Dap, similar to the fluorophore labeling strategy outlined above. NaCNBFL reduction can be performed on the bacterial cell bound peptides. It is suggested that KAM5 binds S. aureus through conjugation with Lys-PG, while KAM8 binds the LOS-A.
  • a successful peptide probe may bind specific surface proteins.
  • genetic tools such as transposon mutagenesis (i.e. Tn-Seq) can be used for target identification. FACS sorting of a transposon library stained by a peptide probe followed by sequencing could inform on the potential target of the probe.
  • an initial set was chosen to include several mechanistically distinct antibiotics (Figure 27).
  • Figure 27 As the peptide probes identified from phage display are likely to bind bacterial cell surfaces without cell entry, the focus is on antibiotics that attack the cell envelope (instead of intracellular targets) for bacterial killing. The ease of conjugation to the peptide probes is used as a secondary criteria.
  • Vancomycin (Dl) has been utilized in hospitals to treat gram-positive infections for several decades. It inhibits bacterial cell growth by binding to the D-Ala-D-Ala dipeptide segment of the lipid II stem peptide, thereby inhibiting peptidoglycan biosynthesis.
  • Daptomycin (D2) another last-resort antibiotic to treat gram-positive infections, is believed to cause bacterial cell death by binding and disrupting the cell membrane of bacteria.
  • Colistin (D3) was introduced half a century ago and had not been a primary antibiotic due to its nephrotoxicity. However, it has recently re-emerged as a last-resort antibiotic for multidrug- resistant gram-negative infections. Colistin is believed to exert its antibiotic activity by binding to lipid A and lipopolysaccharide (LPS) and subsequently disrupting the membrane of the cells.
  • LPS lipopolysaccharide
  • These antibiotics carry an amino group (H2N in Figure 27) that has been shown to be non-essential for function and therefore can serve as a handle for conjugation to a directing element.
  • SMAMP-02 (D4) is a structural analogue of brilacidin, an antimicrobial peptide mimic currently in clinical trials. SMAMP-02 is chosen for the studies because of its ease of conjugation and broad-spectrum activity with potency similar to that of brilacidin.
  • LTX-009 (D5) is another membrane-disrupting antibiotic currently in clinical trials.
  • a clickable handle can be installed onto the primary amines of the bactericidal compounds, which can be used to conjugate with a peptide binder of bacteria.
  • azide-alkyne click chemistry is used to introduce the 2-APBA moiety to the bicyclic peptides ( Figure 26)
  • the tetrazine-based bioconjugation chemistries can be chosen for joining the antibiotic and the peptides.
  • a tetrazine moiety can be installed onto the peptide probes similar to the fluorophore conjugation protocol described earlier.
  • the antibiotics can be derivatized with a linker and a trans-octene group for tetrazine conjugation. This modular design should allow facile incorporation and assessment of both stable and cleavable (-S-S-) linkers in the peptide-antibiotic conjugates.
  • Targeted antibiotics can be developed via conjugation of a bactericidal agent to a directing peptide that binds specific bacterial strains.
  • the peptide-antibiotic conjugates can be first tested in vitro to determine the MIC values for the panel of bacteria described above. In comparison to the parent antibiotics, the peptide-antibiotic conjugates are expected to gain potency (lowered MIC) toward the target strain, while bypassing other bacteria as well as host cells. Comparative analysis of the MICs across the panel of bacteria can inform on the species and strain specificity. Host cell toxicity can be assessed against red blood cells and additional model cell lines.
  • the vancomycin/daptomycin conjugates shows efficacy against gram-positives, while the colistin conjugates works against gram-negative bacteria.
  • the conjugates of the synthetic antibiotics (D4- 6) may show broader applicability with specificity dictated by the directing peptide.
  • the peptide-antibiotic conjugates showing high potency and selectivity in vitro can be further tested in animal models of bacteremia.
  • the in vivo testing can focus on the infections caused by S. aureus and A. baumannii due to the continued high mortality of S. aureus and the increasing severity of drug resistance shown by A. baumannii.
  • For each bacteria two of the top performing peptide-antibiotic conjugates identified from in vitro studies can be tested. Briefly, for S. aureus infection, NMRI mice aged 5-7 weeks are injected in the tail vein with -10 7 CFUs of bacteria (S. aureus ⁇ . ATCC 43300).
  • mice One cohort of ten mice is treated with a peptide-antibiotic conjugate, while the control cohort is injected with the vehicle alone for comparison.
  • the dosage and frequency of drug administration are determined empirically through a separate experiment beforehand. Bacterial loads are determined daily for seven days via tail bleeds and subsequent titering and plating of bacteria on agar. To determine whether the peptide-antibiotic conjugate can increase the survival rate, mice are infected with 3* l0 8 CFUs, which typically causes mortality within 100 hours post infection. The same number of mice and treatment groups are used as in the bacterial load experiments. Mice are euthanized when moribund, bacterial loads are determined and time of death post infection is used to construct Kaplan-Meier survival curves. A. baumannii infection is modeled through a similar protocol except that ICR mice (6-8 weeks) are used. The successful designs of a peptide-antibiotic conjugate can elicit better bacterial clearance and increase the survival rate of the animals.

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Abstract

Plateforme d'affichage de bactériophage chimiquement modifié et son procédé d'utilisation. Plus spécifiquement, la présente invention concerne une bibliothèque de présentation de phages chimiquement modifiés qui renferme des fragments d'acide 2-acétylphénylboronique (APBA) pour obtenir une liaison covalente dynamique à la surface de cellule bactérienne. Les bibliothèques de présentation de bactériophages modifiés par APBA décrites ici sont applicables à un large réseau de souches bactériennes et/ou de cellules de mammifère, ouvrant la voie à un diagnostic et un développement simplistes d'antibiotiques spécifiques à la souche et/ou de conjugués peptides-antibiotiques pour un traitement efficace et ciblé. L'invention concerne également des peptides thérapeutiques, et des compositions pharmaceutiques de ceux-ci, qui sont identifiés par criblage de la bibliothèque de présentation de bactériophages de la présente invention, et un procédé d'utilisation de ces peptides thérapeutiques pour un traitement efficace et ciblé.
PCT/US2019/043211 2018-07-25 2019-07-24 Procédés et compositions de bibliothèques de bactériophages modifiés chimiquement WO2020023620A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040266789A1 (en) * 2003-04-30 2004-12-30 Darren Whitehouse Substituted amino carboxylic acids
US20090148887A1 (en) * 2007-11-02 2009-06-11 The Scripps Research Institute Genetically encoded boronate amino acid
WO2013084198A1 (fr) * 2011-12-07 2013-06-13 Universidade De Lisboa Modification chimique et bioconjugaison de protéines ou de peptides au moyen de composés de bore
US20150166988A1 (en) * 2008-02-05 2015-06-18 Bicycle Therapeutics Limited Methods and compositions
US20150329568A1 (en) * 2014-04-23 2015-11-19 The Research Foundation For The State University Of New York Rapid and efficient bioorthogonal ligation reaction and boron-containing heterocycles useful in conjunction therewith

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040266789A1 (en) * 2003-04-30 2004-12-30 Darren Whitehouse Substituted amino carboxylic acids
US20090148887A1 (en) * 2007-11-02 2009-06-11 The Scripps Research Institute Genetically encoded boronate amino acid
US20150166988A1 (en) * 2008-02-05 2015-06-18 Bicycle Therapeutics Limited Methods and compositions
WO2013084198A1 (fr) * 2011-12-07 2013-06-13 Universidade De Lisboa Modification chimique et bioconjugaison de protéines ou de peptides au moyen de composés de bore
US20150329568A1 (en) * 2014-04-23 2015-11-19 The Research Foundation For The State University Of New York Rapid and efficient bioorthogonal ligation reaction and boron-containing heterocycles useful in conjunction therewith

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Title
DIEMER ET AL.: "Simultaneous Disulfide and Boronic Acid Ester Exchange in Dynamic Combinatorial Libraries", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 16, no. 9, 10 September 2015 (2015-09-10), pages 21858 - 21872, XP055272013, DOI: 10.3390/ijms160921858 *
MCCARTHY ET AL.: "Phage Display of Dynamic Covalent Binding Motifs Enables Facile Development of Targeted Antibiotics", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 140, no. 19, 27 April 2018 (2018-04-27), pages 6137 - 6145, XP055682341, DOI: 10.1021/jacs.8b02461 *

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