Described herein are methods of physicochemical-guided peptide design that utilize specific functional determinants to a protein's property of interest. Also described herein are novel synthetic peptide antibiotics that have increased potency and/or decreased toxicity relative to the template peptide from which they were derived, and methods of use thereof in treating microbial infections.
Drug-resistant bacteria are a major health problem worldwide (CDC Current. 114, doi: CS239559-B (2013)). Even in developed countries such as the United States, each year ˜2 million people become infected with antibiotic-resistant bacteria, resulting in at least 23,000 deaths annually (CDC Current. 114, doi:CS239559-B (2013)). Therefore, there is an urgent need to develop new therapeutics to combat drug resistance (Walsh C., Nature. 406, 775-781 (2000); Arora G. et al., Springer. doi:10.1007/978-3-319-48683-3 (2017)).
Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics because of their potency against difficult-to-treat infections (Mahlapuu M., et al., Front. Cell. Infect. Microbiol. 6, 1-12 (2016)), such as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) (Pendleton J. N., et al., Expert Rev. Anti. Infect. Ther. 11, 297-308 (2013)), which are relevant microorganisms for posing a clinical threat for the existing treatments due to their virulence, resistance, transmission and pathogenicity. AMPs are produced as a mechanism of defense (e.g., against infections) by virtually all living organisms. Some of these peptides exhibit broad-spectrum activity, targeting both bacterial and mammalian cells indiscriminately. However, the biological function of AMPs may be tuned by modulating biophysical features to favor specificity, selectivity (de la Fuente-Nunez C. et al., Curr. Opin. Microbiol. 37, 95-102 (2017)), potency (Melo M. N. et al., Nat. Rev. Microbiol. 7, 245-250 (2009)) and other desired biological parameters to turn these molecules into novel anti-infective agents.
As described herein, a rational peptide design strategy aimed at tuning physicochemical features involved in structure and function such as hydrophobicity, net positive charge, and helical content, was used to generate novel peptide antibiotics.
In some aspects, the disclosure relates to antimicrobial peptides. In some embodiments, the antimicrobial peptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-383. In some embodiments, the antimicrobial peptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21. In some embodiments, the antimicrobial peptide consists of the amino acid sequence of any one of SEQ ID NOs: 2-383. In some embodiments, the antimicrobial peptide consists of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21.
In some aspects, the disclosure relates to compositions comprising an antimicrobial peptide described herein (e.g., comprising the amino acid sequence of any one of SEQ ID NOs: 2-383). In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
In yet other aspects, the disclosure relates to methods of treating a microbial infection. In some embodiments, the method comprises administering, to a subject in need of such treatment, a therapeutically effective amount of an antimicrobial peptide described herein (e.g., comprising the amino acid sequence of any one of SEQ ID NOs: 2-383). In other embodiments, the method comprises administering, to a subject in need of such treatment, a therapeutically effective amount of a composition described herein (e.g., comprising an antimicrobial peptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-383).
In some embodiments, the subject is a mammal. In some embodiments, the subject is human.
In some embodiments, the antimicrobial peptide or the composition is administered orally, intravenously, intramuscularly, subcutaneously, or topically.
In some embodiments, the microbial infection comprises a bacterial, fungal, algal, viral, or protozoan infection.
In some embodiments, the microbial infection comprises a bacterial infection.
In some embodiments, the bacterial infection comprises a Gram-positive bacterium. For example, in some embodiments, the bacterial infection comprises a bacterium selected from the group consisting of a Micrococcus luteus bacterium, a Staphylococcus aureus bacterium, a Staphylococcus epidermidis bacterium, a Bacillus megaterium bacterium, and an Enterococcus faecium bacterium. In some embodiments: (a) the bacterium is a Micrococcus luteus bacterium, wherein the Micrococcus luteus bacterium is strain A270; (b) the bacterium is a Staphylococcus aureus bacterium, wherein the Staphylococcus aureus bacterium is strain ATCC29213 or ATCC12600; (c) the bacterium is a Staphylococcus epidermidis bacterium, wherein the Staphylococcus epidermidis bacterium is a strain ATCC12228; or (d) the bacterium is a Bacillus megaterium bacterium, wherein the Bacillus megaterium bacterium is a strain ATCC10778.
In some embodiments, the bacterial infection comprises a Gram-negative bacterium. For example, in some embodiments, the bacterial infection comprises a bacterium selected from the group consisting of an Escherichia coli bacterium, an Enterobacter cloacae bacterium, a Serratia marcescens bacterium, a Pseudomonas aeruginosa bacterium, a Klebsiella pneumoniae bacterium, and an Acinetobacter baumannii bacterium. In some embodiments: (a) the bacterium is an Escherichia coli bacterium, wherein the Escherichia coli bacterium is a strain SBS 363 or BL21; (b) the bacterium is an Enterobacter cloacae bacterium, wherein the Enterobacter cloacae bacterium is a strain ®-12; (c) the bacterium is a Serratia marcescens bacterium, wherein the Serratia marcescens bacterium is a strain ATCC4112; or (d) the bacterium is a Pseudomonas aeruginosa bacterium, wherein the Pseudomonas aeruginosa bacterium is a strain PA14 or PA01.
In some embodiments, the microbial infection comprises a fungal infection. In some embodiments, the fungal infection comprises a pathogenic yeast. For example, in some embodiments, the fungal infection comprises a pathogenic yeast selected from the group consisting of Candida albicans and Candida tropicalis. In some embodiments: (a) the pathogenic yeast is Candida albicans, wherein the Candida albicans is strain MDM8; or (b) the pathogenic yeast is Candida tropicalis, wherein the Candida tropicalis is strain IOC4560.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.
Despite some obstacles, such as short half-life in blood stream-like environments of small linear natural peptides and intrinsic bacterial resistance (i.e. membrane modifications efflux and proteolytic degradation) to certain host defense peptides (Andersson D. I. et al, Drug Resist. Updat. 26, 43-57 (2016)), AMPs are a promising alternative to conventional antibiotics because of their unique diversity of peptide sequences. Their sequence space is almost unlimited, and a wide range of amino acids is available in nature (Perumal Samy R. et al., Biochem. Pharm. 134, (2017)). Biological evolution has selected AMPs with certain sequence biases; however, even minor changes to these sequences enabled by peptide engineering may yield unprecedented biological function. The most widely studied class of AMPs is that comprising the linear cationic amphipathic AMPs (Hancock R. E. W. Expert Opin. Investig. Drugs 9, 1723-1729 (2000)), which shift from coiled to helical structures (Lifson S. and Roig A. J. Chem. Phys. 34, 1963-1974 (1961); Zimm B. H. and Bragg J. K. J. Chem. Phys. 31, 526-535 (1959)) when the peptide comes into contact with membranes of microorganisms.
Most AMPs act by disrupting the cytoplasmic membrane of microorganisms in ways (Nguyen L.T. et al., Trends Biotechnol. 29, 464-472 (2011)) that are not necessarily exclusive of one another. Important mechanisms of action of AMPs are carpet-like, barrel stave, or toroidal pore formation (Brogden K. A. Nat. Rev. Microbiol. 3, 238 (2005)). Other specific or general mechanisms have been described, such as membrane thickening/thinning (Lohner K. Gen. Physiol. Biophys. 28, 105-116 (2009)), charged lipid clustering (Epand R. M. and Epand R. F. J. Pept. Sci. 17, 298-305 (2011)), nucleic acids targeting (Brogden K. A. Nat. Rev. Microbiol. 3, 238 (2005)), anion carriers (Rokitskaya T. I. et al., Biochim. Biophys. Acta—Biomembr. 1808, 91-97 (2011)), electroporation (Chan D. I. et al., Biochim. Biophys. Acta—Biomembr. 1758, 1184-1202 (2006)), non-lytic membrane depolarization (Gifford J. L. et al., Cell. Mol. Life Sci. 62, 2588-98 (2005)), and non-bilayer intermediates (Haney E. F. et al., Chem. Phys. Lipids 163, 82-93 (2010)). However, some AMPs antimicrobial mode of action include targeting key cellular processes and metabolic pathways (Le C. F. et al., Sci. Rep. 6, 26828 (2016); Huang N. et al., Tumor Biol. 39, 1010428317708532 (2017)) including DNA and protein synthesis (Park C. B. et al., Biochem. Biophys. Res. Commun. 244, 253-257 (1998); Krizsan A. et al., Eur. J. Chem. Biol. 16, 2304-2308 (2015)), protein folding, enzymatic activity and cell wall synthesis (de Kruijff B. et al., Prostaglandins, Leukot. Essent. Fat. Acids 79, 117-121 (2008)), cell division (Subbalakshmi C. and Sitaram N. FEMS Microbiol. Lett. 160, 91-96 (1998)), RNA synthesis (Haney E. F. et al., Biochim. Biophys. Acta—Biomembr. 1828, 1802-1813 (2013)), inactivation of chaperone proteins necessary for proper folding, and even targeting mitochondria (da Costa J. P. et al., Appl. Microbiol. Biotechnol. 99, 2023-2040 (2015)).
Insects such as wasps, scorpions and spiders are rich sources of linear cationic amphipathic AMPs (Perumal Samy R. et al., Biochem. Pharm. 134, (2017)). The South American social wasp Polybia paulista has a large variety of peptides in its venom, each of which has a different biological function (Lee S. H. et al., Toxins (Basel). 8, 1-29 (2016)). Among them, the mastoparan class is a well-known group of chemotactic peptides having inflammatory and antimicrobial activities (Lee S. H. et al., Toxins (Basel). 8, 1-29 (2016)). Souza et al. reported a 12-residue cationic amphipathic mastoparan-like AMP, polybia-CP (Pol-CP—NH2: Ile-Leu-Gly-Thr-Ile-Leu-Gly-Leu-Leu-Lys-Ser-Leu-NH2 (SEQ ID NO: 1)), which presents poor activity against Gram-negative bacteria, higher activity against Gram-positive bacteria, and toxicity towards human cells (Souza B. M. et al., Peptides 26, 2157-2164 (2005)). The lower activity of Pol-CP—NH2 against Gram-negative bacteria was attributed to its low predicted helical content and to the presence of a hydrophilic serine residue next to its C-terminus, a residue that is not present in this position in other mastoparan-like peptides from the same wasp venom, such as protonectin and polybia-MPI (Souza B. M. et al., Peptides 26, 2157-2164 (2005)).
As described herein, a rational peptide design strategy aimed at tuning physicochemical features involved in structure and function such as hydrophobicity, net positive charge, and helical content, was used herein to improve the antimicrobial activity of Pol-CP—NH2 and to generate novel peptide antibiotics (
As such, in some aspects, the disclosure relates to synthetic antimicrobial peptides (i.e., consisting of an amino acid sequence that is not found in nature). In some embodiments, the antimicrobial peptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-383. In some embodiments, the antimicrobial peptide comprises the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21. In some embodiments, the antimicrobial peptide consists of the amino acid sequence of any one of SEQ ID NOs: 2-383. In some embodiments, the antimicrobial peptide consists of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 21.
In some aspects, the disclosure relates to compositions comprising an antimicrobial peptide described herein (e.g., an antimicrobial peptide comprising or consisting of any one of SEQ ID NOs: 2-383).
In some embodiments, each of the antimicrobial peptides in the composition are chemically identical.
In other embodiments, the composition comprises a plurality of antimicrobial peptides comprising chemically distinct antimicrobial peptides. For example, in some embodiments, a composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more chemically distinct antimicrobial peptides. In some embodiments, a composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 chemically distinct antimicrobial peptides. In some embodiments, a composition comprises 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-25, 10-30, 10-35, 10-40, 10-45, or 10-50 chemically distinct antimicrobial peptides.
In some embodiments, each of the antimicrobial peptides in the composition comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 2-383. In some embodiments, a subset of the antimicrobial peptides in the composition comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 2-383.
The compositions described herein may further comprise a pharmaceutically-acceptable carrier. Generally, for pharmaceutical use, the composition may be formulated as a pharmaceutical preparation or composition comprising at least one active unit (i.e., at least one antimicrobial peptide) and at least one pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active compounds. Such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such administration forms may be solid, semi-solid or liquid, depending on the manner and route of administration. For example, formulations for oral administration may be provided with an enteric coating that will allow the formulation to resist the gastric environment and pass into the intestines. More generally, formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract. Various pharmaceutically acceptable carriers, diluents and excipients useful in therapeutic compositions are known to the skilled person.
As used herein, the term “pharmaceutically-acceptable carrier” refers to one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other subject contemplated by the disclosure. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers (e.g., antioxidants), gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
In yet other aspects, the disclosure relates to methods of treating a microbial infection in a subject in need of such a treatment. In some embodiments the subject is a mammal, such as a human.
In some embodiments, the method comprises administering, to a subject in need of such treatment, a therapeutically effective amount of an antimicrobial peptide as described herein (or a composition comprising an antimicrobial peptide as described herein). The antimicrobial peptide (or composition comprising an antimicrobial peptide) may be administered orally, intravenously, intramuscularly, subcutaneously, or topically. Alternatively or in addition, administration may be by inhalation, by a skin patch, by an implant, by a suppository, etc. Additional modes of administration are known to those having ordinary skill in the art.
As used herein the term “therapeutically effective” applied to an amount refers to that quantity of a compound or composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. For example, the term “therapeutically effective” refers to a quantity of a compound or pharmaceutical composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom associated with a microbial infection.
A microbial infection may comprise a bacterial infection, a fungal infection, an algal infection, a viral infection, a protozoan infection, or a combination thereof. Examples of bacterial infections, fungal infections, algal infections, viral infections, and protozoan infections are known to those having ordinary skill in the art.
In some embodiments, a microbial infection comprises a bacterial infection.
The bacterial infection may comprise a Gram-positive bacterium, such as a Micrococcus luteus bacterium, a Staphylococcus aureus bacterium, a Staphylococcus epidermidis bacterium, a Bacillus megaterium bacterium, or an Enterococcus faecium bacterium. Additional infectious Gram-positive bacterium are known to those having ordinary skill in the art. In some embodiments, a bacterial infection comprises: (a) a Micrococcus luteus bacterium, wherein the Micrococcus luteus bacterium is strain A270; (b) a Staphylococcus aureus bacterium, wherein the Staphylococcus aureus bacterium is strain ATCC29213 or ATCC12600; (c) a Staphylococcus epidermidis bacterium, wherein the Staphylococcus epidermidis bacterium is a strain ATCC12228; (d) a Bacillus megaterium bacterium, wherein the Bacillus megaterium bacterium is a strain ATCC10778; or (e) a combination thereof.
Alternatively or in addition, the bacterial infection may comprise a Gram-negative bacterium, such as an Escherichia coli bacterium, an Enterobacter cloacae bacterium, a Serratia marcescens bacterium, a Pseudomonas aeruginosa bacterium, a Klebsiella pneumoniae bacterium, or an Acinetobacter baumannii bacterium. Additional infectious Gram-negative bacterium are known to those having ordinary skill in the art. In some embodiments, a bacterial infection comprises: (a) an Escherichia coli bacterium, wherein the Escherichia coli bacterium is a strain SBS 363 or BL21; (b) an Enterobacter cloacae bacterium, wherein the Enterobacter cloacae bacterium is a strain ®-12; (c) a Serratia marcescens bacterium, wherein the Serratia marcescens bacterium is a strain ATCC4112; (d) a Pseudomonas aeruginosa bacterium, wherein the Pseudomonas aeruginosa bacterium is a strain PA14 or PA01; or (e) a combination thereof.
Alternatively or in addition, a microbial infection may comprise a fungal infection. The fungal infection may comprise a pathogenic yeast, such as Candida albicans or Candida tropicalis. Additional pathogenic yeast are known to those have ordinary skill in the art. In some embodiments, the fungal infection comprises: (a) Candida albicans, wherein the Candida albicans is strain MDM8; (b) Candida tropicalis, wherein the Candida tropicalis is strain IOC4560; or (c) a combination thereof.
Solid-phase peptide synthesis (SPPS), Purification and Analysis. Ala-scan analogs were acquired from Biopolymers and the second generation of peptides was synthesized on a peptide synthesizer (PS3—Sync Technologies) using the fluoromethyloxycarbonyl (Fmoc) strategy, Rink Amide resin, with a substitution degree of 0.52 mmol g−1. Deprotection steps were carried out by treatment with 4-methylpiperidine in dimethylformamide (4-MePip/DMF, 1:4, v/v) for 40 minutes. Amino acid coupling steps were accomplished by treating the deprotected amino acyl-resin with 4-fold molar excess of the Fmoc-protected amino acid, activated by N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uranium hexafluorophosphate (HBTU) in DMF, for 30 minutes at room temperature. Each step was followed by a washing procedure with DMF to favor resin swelling, elimination of excess reagents and byproducts, leading to the peptidyl-resin.
Dry-protected peptidyl-resin was exposed to trifluoroacetic acid (TFA)/Anisole/Water (95:2.5:2.5, v/v/v) for two hours at room temperature. The crude deprotected peptides were precipitated with anhydrous diethyl ether, filtered from the ether-soluble products, extracted from the resin with 60% ACN (acetonitrile) in water and lyophilized.
The crude lyophilized peptides were then purified by preparative reverse-phase high-performance liquid chromatography (RP-HPLC) in 0.1% TFA/90% ACN in water (A/B) on a Delta Prep 600 (Waters Associates). Briefly, the peptides were loaded onto a Phenomenex C18 (21.2 mm×250 mm, 15 μm particles, 300 Å pores) column at a flow rate of 10.0 mL min−1 and eluted using a linear gradient (0.33% B/min slope), with detection at 220 nm. Selected fractions containing the purified peptides were pooled and lyophilized. Purified peptides were characterized by liquid-chromatography electrospray-ionization mass spectrometry (LC/ESI-MS).
LC/ESI-MS data were obtained on a Model 6130 Infinity mass spectrometer coupled to a Model 1260 HPLC system (Agilent) using a Phenomenex Gemini C18 column (2.0 mm×150 mm, 3.0 μm particles, 110 Å pores). Solvent A was 0.1% TFA in water, and solvent B was 90% ACN in solvent A. Elution with a 5-95% B gradient was performed over 20 min, 0,2 mLmin−1 flow and peptides were detected at 220 nm. Mass measurements were performed in a positive mode with the following conditions: mass range between 100 to 2500 m/z, ion energy of 5.0 V, nitrogen gas flow of 12 L min−1, solvent heater of 250° C., multiplier of 1.0, capillary of 3.0 kV and cone voltage of 35 V.
Circular dichroism (CD) spectroscopy. CD experiments were performed on a J-815 Circular Dichroism Spectropolarimeter (Jasco). Far-UV CD spectra were recorded after four accumulations at 20° C., using a 1 mm path length quartz cell, between 260 and 190 nm at 50 nm min−1 with a band width of 0.5 nm. All peptides were analyzed in water, PBS (pH 7.4), MeOH/water (1:1; v:v), TFE/Water (2:3, 3:2 and 4:1; v:v), 10 mmol L−1 POPC, 10 mmol L−1 POPC:DOPE (3:1) and 10 mmol L−1 POPC:POPG (3:1). The large unilamellar vesicles (POPC, POPC:DOPE and POPC:POPG) preparation was by the formation of a lipid film of the desired composition on the walls of a test tube from a lipid stock solution in chloroform, dried with a stream of N2 and kept in a vacuum for 1 h. The lipid film was resuspended in a buffer solution (10 mmol L−1 PBS, pH 7.4), and vortexed to form multilamellar vesicles. This lipid dispersion was extruded at least 21 times through a polycarbonate membrane with a pore size of 100 nm to yield the large unilamellar vesicles. The peptide concentration was 50 mol L−1. A Fourier transform filter was applied to minimize background effects.
Microorganisms. The following strains were used: Micrococcus luteus A270, Staphylococcus aureus ATCC29213, Staphylococcus epidermidis ATCC12228, Bacillus megaterium ATCC10778 Escherichia coli SBS 363, Enterobacter cloacae ®-12, Serratia marcescens ATCC4112, Candida albicans MDM8, Candida tropicalis IOC4560 from Instituto Butanta, Sao Paulo, Brazil, and Escherichia coli BL21, Pseudomonas aeruginosa PA14, Pseudomonas aeruginosa PA01 and Staphylococcus aureus ATCC12600 from Synthetic Biology Group at MIT.
MIC assays. The MIC assays were performed using the broth microdilution method (Wiegand I. et al., Protoc. 3, 163-175 (2008); de la Fuente-Núñez C. et al., Antimicrob. Agents Chemother. 56, 2696-2704 (2012)) in sterile 96-well polypropylene microtiter plates. Peptides were added to the plate as solutions in BM2 minimal medium in concentrations ranging from 0 to 128 mol L−1, and the bacteria were inoculated at a final concentration of 5×105 CFU mL−1 per well. The plates were incubated at 37° C. for 24 h. The MIC was defined as the lowest concentration of compound at which no growth was observed. Additional liquid growth inhibition assays were done in Peptone Broth (PB, 0.5% NaCl, 1% Peptone at pH 7.4) and Potato Dextrose Broth (Invitrogen) were used for antibacterial and antifungal assays, respectively. Briefly, bacteria or fungi were incubated with serial dilutions of polybia-CP and analogs (50-0.09 μmol L−1) in a 96-well microplate at 37° C. The microbial growth was assessed by measurements in a model 354 Multiskan Ascent microplate reader at A595nm, after 18 and 24 h (bacteria and fungi, respectively) incubation on a model 347CD FANEM incubator. MIC was defined as the minimal inhibitory concentration that prevents 100% of the bacterial growth. All assays were done in triplicate.
Bacterial Killing Experiments. Killing experiments involved performing 1:10,000 dilutions of overnight cultures of E. coli BL21, S. aureus ATCC12600, P. aeruginosa PA01 and PA14 in the absence or presence of increasing concentrations of Pol-CP—NH2 derivatives (0-64 mol L−1). After 24 h of treatment, 10-fold serial dilutions were performed, bacteria were plated on LB agar plates (E. coli BL21 and S. aureus ATCC12600) and Pseudomonas Isolation Agar (P. aeruginosa PA01 and PA14) and allowed to grow overnight at 37° C. after which colony forming unit (CFU) counts were recorded, according to Wiegand et al. (Wiegand I. et al., Protoc. 3, 163-175 (2008)).
Hemolytic Activity Assays. Human erythrocytes were collected and washed three times by centrifugation at 300×g with PBS (pH 7.4). After the last centrifugation, the cells were resuspended in PBS pH 7.4. Aliquots at a concentration of 0.1 to 100 mol L−1 of the peptides were added to the 96-well microplate, where in each well containing 50 L of a suspension of erythrocytes to 0.4% in a phosphate saline buffer (10 mmol L−1 Na2HPO4, 1.8 mmol L−1 K2HPO4, pH 7.4, 137 mmol L−1 NaCl and 2.7 mmol L−1KCl). After that, the samples were incubated at room temperature for 1 h. Hemolysis was determined by reading absorbance at 405 nm of each well in a bed of plates. 1% SDS in PBS solution was used as positive control (Shalel S. et al., J. Colloid Interface Sci. 252, 66-76 (2002); Love L. J. Cell. Comp. Physiol. 36, 133-148 (1950)) and as negative control was used PBS only. MHC was defined as the maximal non-hemolytic concentration.
Stability Assays. The stability assay was performed with GIBCO fetal bovine serum diluted to 25% in water. 20 L of a 10 mg mL−1 peptide solution was added to 1 mL 25% serum solution and kept at 37° C. The experiments were made in triplicate and 100 L aliquots were taken at 0, 0.5, 1, 2, 4 and 6 h. 10 L of TFA was added to the aliquots and the new solution was kept at 5° C. for 10 min, after that it was centrifuged at 14,000 rpm for 15 min, according to Powell et al. (Powell M. F. et al., Pharm. Res. 10, 1268-1273 (1993)). The reaction kinetics was followed by liquid chromatography and the percentage of remaining peptide was calculated by integrating the peptide peak area.
Cytotoxicity assays. Human embryonic kidney 293 (HEK 293) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin at 37° C. in 5% CO2. The day before treatment, 50,000 HEK 293 cells were seeded into each well in 96-well plates. The peptides were added at concentrations ranging from 0 to 64 mol L−1 and 48 h after exposure, cell viability was measured by means of MTS (dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium) assay. Experiments were performed in triplicate for each condition.
Scarification Skin Infection Mouse Model. P. aeruginosa strain PA14 was grown to an optical density at 600 nm (OD600) of 1 in tryptic soy broth (TSB) medium. Subsequently cells were washed twice with sterile PBS (pH 7.4, 13,000 rpm for 1 minute), and resuspended to a final concentration of 5×107 CFU/50 L. To generate skin infection, female CD-1 mice (6 weeks old) were anesthetized with isoflurane and had their backs shaved. A superficial linear skin abrasion was made with a needle in order to damage the stratum corneum and upper-layer of the epidermis. Five minutes after wounding, an aliquot of 50 L containing 5×107 CFU of bacteria in PBS was inoculated over each defined area containing the scratch with a pipette tip. One day after the infection, peptides were administered to the infected area. Animals were euthanized and the area of scarified skin was excised two and four days post-infection, homogenized using a bead beater for 20 minutes (25 Hz), and serially diluted for CFU quantification. Two independent experiments were performed with 4 mice per group in each condition. Statistical significance was assessed using a one-way ANOVA.
Molecular Modeling. Molecular modeling studies were carried out according to four successive steps: (i) selection of a template structure; (ii) alignment between the template and target sequences; (iii) construction of atomic coordinates; and (iv) validation of the lowest free energy theoretical models. Initially, Blastp was performed and a fragment from the structure of a methyltransferase (chain A) (PDB entry: 3SSM) (Akey D. L. et al., J. Mol. Biol. 413, 438-450 (2011)) was select as template, taking into account parameters such as identity, coverage and e-value. All target sequences were individually aligned to the template and further submitted to comparative modeling simulations on MODELLER v. 9.17 (Fiser A. and Šali A. Academic Press. 374, 461-491 (2003)). A total of 100 models were generated for each peptide and ranked according to their free energy scores (DOPE score). The lowest free energy models for each peptide were validated regarding their stereochemistry and fold quality on PROCHECK (Laskowski R. A. et al., J. Appl. Crystallogr. 26, 283-291 (1993)) and ProSA-web servers (Wiederstein M. and Sippl M. J. Nucleic Acids Res. 35, W407-W410 (2007)). Finally, the validated structures were visualized and analyzed using PyMOL v. 1.8 (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).
Molecular Dynamics. Molecular dynamics simulations were conducted in hydrophilic environment (water) and in a mixture of 60% TFE/water (v/v). The GROMOS 43a1 force field (Lindahl E. et al., Mol. Model. Annu. 7, 306-317 (2001)) was used and the simulation and analysis performed using the computational package GROMACS 5.0.4 (Abraham M. J. et al., SoftwareX 1-2, 19-25 (2015)). As initial structures, the validated models obtained from molecular modeling simulations were immersed in cubic boxes containing single point charge (SPC) water molecules. Simulations in 60% TFE were also performed in cubic boxes, the peptides immersed in SPC water molecules, followed by the insertion of TFE molecules until the ideal concentration was reached. Chloride ions (Cl−) were also added to neutralize the system's charge. Moreover, the LINCS algorithm was used to link all the atom bond length. Particle Mesh Ewald (PME) was also used for electrostatic corrections, with a radius cut-off of 1.4 nm to minimize the computational simulation time. The same radius cut off was also used for van der Waals interactions. The list of neighbors of each atom was updated every 10 simulation steps of 2 fs each. A conjugate gradient (2 ns) and the steepest descent algorithms (2 ns) were implemented for energy minimization. After that, the systems underwent a normalization of pressure and temperature, using the integrator stochastic dynamics, 2 ns each. The systems with minimized energy and balanced temperature and pressure was carried out using a step of position restraint, using the integrator Molecular Dynamics (MD), for 2 ns. The simulations were carried out during 100 ns at 27° C. in silico, aiming to understand the structural conformation of the peptide more nearly to that observed in vitro bioassays. All simulations were programmed in triplicate.
The first generation of peptides was designed to evaluate the role of the side chain of each residue in biological function, and to determine how substitutions to the side chain groups of each residue would alter structural and physicochemical features when compared to those of the helical wild-type peptide Pol-CP—NH2. Because Ala presents the smallest side chain among all-natural chiral amino acids, it was chosen to conserve the backbone size and to evaluate the effect of the native side chains on both structure and activity.
First, theoretical values of physicochemical features such as hydrophobicity, hydrophobic moment, and net positive charge were calculated, and helical wheels were generated using the Heliquest webserver (Gautier R. et al., Bioinformatics 24, 2101-2102 (2008)) (
Substitution analysis with Ala revealed that when Ile at position 5 and, independently, Lys at position 10 were substituted, the most drastic decreases in antimicrobial activity were observed against both Gram-positive and Gram-negative bacteria (
To further investigate the effect of side chains on the structure of Pol-CP—NH2, circular dichroism (CD) spectroscopy measurements were performed, a rapid and widely used technique for analyzing peptides secondary structure, which is determinant for AMPs activity (Greenfield N. J. Trends Anal. Chem. 18, 236-244 (1999)). Among all the features that can be extracted from CD analyses, of particular interest were potential structure transitions, more specifically helix-coil transition usually observed from water or polar media to hydrophobic or helical inducer-media (Lifson S. and Roig A. J. Chem. Phys. 34, 1963-1974 (1961)), a very well-known characteristic of AMPs (Pedron C. N. et al., Eur. J. Med. Chem. 126, 456-463 (2017); Tones M. D. T. et al., ChemistrySelect 2, 18-23 (2017); Tones M. D. T. et al., J. Pept. Sci. 23, 818-823 (2017); Zelezetsky I. and Tossi A. Biochim. Biophys. Acta—Biomembr. 1758, 1436-1449 (2006); Porto W. F. et al., Nat. Commun. 9, 1490 (2018)). For this reason, the experiments were performed initially in three conditions [i.e., water, PBS buffer (pH 7.4), and trifluoroethanol (TFE) in water (3:2; v:v)] using the Ala-scan derivatives. The PBS buffer was chosen to check the effects of peptides exposure to ions at neutral pH (7.4), besides it low absorbance at the wavelength range analyzed (195 to 260 nm). TFE/water solution is widely used in studies of peptide structure as it promotes the formation of helical structures and stability (Buck M. Q. Rev. Biophys. 31, 297-355 (1998); Luo P. and Baldwin R. L. Biochemistry 36, 8413-8421 (1997)). As expected, the peptides presented an undefined secondary structure in water and a secondary structure with small helical fractions in PBS buffer (saline environment). In contrast, in the presence of TFE/water solution, the peptides tended to display a helical structure (
Molecular dynamics (MD) simulations of the peptides were performed in water and in 60% TFE/water solution (v:v). The simulations were performed to better understand the behavior of the three-dimensional theoretical structure (
aLC/ESI-MS data were obtained on a Model 6130 Infinity mass spectrometer coupled to a Model 1260 HPLC system (Agilent), using a Phenomenex Gemini C18 column (2.0 mm × 150 mm, 3.0 μm particles, 110 Å pores). Solvent A was 0.1% TFA in water, and solvent B was 90% ACN in solvent A. Elution with a 5-95% B gradient was performed over 20 min, 0.2 mL min−1 flow and peptides were detected at 220 nm. Mass measurements were performed in a positive mode with the following conditions: mass range between 100 to 2500 m/z, ion energy of 5.0 V, nitrogen gas flow of 12 L min−1, solvent heater of 250° C., multiplier of 1.0, capillary of 3.0 kV and cone voltage of 35 V.
b, cHPLC profiles were obtained under the following conditions: Column Supelcosil C18 (4.6 × 150 mm), 60 Å, 5 μm; Solvent System: A (0.1% TFA/H2O) and B (0.1% TFA in 90% ACN/H2O); Gradient: 5-95% B in 30 minutes; Flow: 1.0 mL min−1; λ = 220 nm; Injection Volume: 50 μL and Sample Concentration: 1.0 mg mL−1.
dConcentration needed for 50% hemolysis caused by RBC exposure to peptides.
eSelectivity Index = HC50/MICaverage indicating peptides selectivity when in the presence of human erythrocytes.
Most wasp venom peptides present conserved motifs in their sequences, e.g., Pol-CP—NH2 is similar to protonectin (Ile-Leu-Gly-Thr-Ile-Leu-Gly-Leu-Leu-Lys-Gly-Leu-NH2 (SEQ ID NO: 385)) (Mendes M. A. et al., Toxicon 44,67-74 (2004)). Therefore, to design the next generation of Pol-CP—NH2 derivatives, single-substitution mutants were generated to elucidate structure-function relationships and to identify physicochemical activity determinants (
To introduce charge into the sequence (Pace C. N. and Scholtz J. M. Biophys. J. 75, 422-427 (1998)), Lys was used rather than Arg due to its superior flexibility, lower propensity in potentially toxic cell penetrating peptides (Cutrona K. J. et al., FEBS Lett. 589, 3915-3920 (2015)), and decreased hydrophobic side chain, which is associated with cytotoxicity (Eisenberg D. Ann. Rev. Biochem. 53,595-623 (1984)). Moreover, Lys residues are more frequent than Arg residues in naturally occurring wasp venom peptides (Lee S. H. et al., Toxins (Basel). 8, 1-29 (2016)).
Hydrophobicity was incorporated into the sequence via substitution of residues from the wild-type sequence by Leu and Phe. Leu was chosen because a minimal amount of energy is required for it to adopt a helical structure (Pace C. N. and Scholtz J. M. Biophys. J. 75,422-427 (1998)), which favors antimicrobial activity (
First, the effects of each substitution on the theoretical values specific physicochemical features was assessed (
Next, peptides were tested against a larger panel of Gram-positive and Gram-negative bacteria and two species of Candida (
MD simulations were performed in water and 60% TFE/water solution (v:v) (
The hemolytic activity of AMPs directly correlates with their interaction with zwitterionic membranes, which they subsequently lyse (Jin Y. et al., ntimicrob. Agents Chemother. 49, 4957-4964 (2005)). Tuning AMP features to modulate membrane interactions to minimize their effect on erythrocyte membranes while preserving activity against bacteria is a long-standing goal in the field. One of the most important parameters to achieve this selectivity is tuning the electronic density—positively charged surfaces—of AMPs, which are attracted to the negatively charged membranes of microorganisms, whereas eukaryotic cells display zwitterionic lipids in their membrane (Lohner K. Norfolk: Horizon Scientific Press. 149-165 (2001)). Mammalian cells present higher amounts of cholesterol in their membrane, which stabilizes the lipid bilayer by increasing cohesion and mechanical stiffness (Henriksen J. et al., Biophys. J. 90, 1639-1649 (2006)), making it difficult for the membranes to bend and, consequently, to be permeabilized by AMPs. After the initial electrostatic interactions, the hydrophobic face of the amphipathic structure of AMPs interacts directly with the nonpolar region of the microorganism membrane, destabilizing it and leading to membrane disruption and cell death (Nagaraj N. S. Curr. Pharm. Des. 8, 727-742 (2002); Yeaman M. R. and Yount N. Y. Pharmacol. Rev. 55, 27 LP-55 (2003)). The design methodology focused primarily in enhancing features that would increase peptides interaction with negatively charged membranes. Thus, the hemolytic activity (
Stability is an issue often limiting the translation of AMPs into the clinic (Seo M. D. et al., Molecules 17, 12276-12286 (2012)). Pol-CP—NH2 is a natural occurring cationic AMP, and most cationic peptides are not stable in the presence of peptidases (Diao L. and Meibohm B. Clin. Pharmacokinet. 52, 855-868 (2013)). The stability of the second generation of Pol-CP—NH2 derivatives (
Several peptides from both generations identified as most active (i.e., antimicrobial hits) and least active (i.e., negative controls) against the Gram-negative bacterium P. aeruginosa (
A skin abscess was induced in mice, after which a single dose of 4 μmol L−1 of peptides was administered (
Additional K7-Polybia-CP—NH2 variants were identified for future analysis using ILGTILKLLKSL (SEQ ID NO: 17) as template, as well as L10-Decoralin-NH2 variants using SLLSLIRKLLT (SEQ ID NO: 22) as template (TABLE 2). For K7-Polybia-CP—NH2 variants, changes at positions 5 (i.e., [Ile]5) and 7 (i.e., [Lys]7) result in sharp drops in activity, and for L10-Decoralin-NH2 variants, changes at positions 8 (i.e., [Lys]8) and 10 (i.e., [Leu]10) result in sharp drops in activity. D-amino analogs and cyclic analogs (synthesized containing the restriction in positions 0 and 13 (CILGTILKLLKSLC; SEQ ID NO: 384) for K7-Polybia-CP—NH2 variants and positions 0 and 12 (CSLLSLIRKLLTC; SEQ ID NO: 385) for L10-Decoralin-NH2 variants) will be explored as well.
AMPs represent promising alternatives to conventional antibiotics to combat the global health problem of antibiotic resistance (Mahlapuu M., et al., Front. Cell. Infect. Microbiol. 6, 1-12 (2016); de la Fuente-Nunez C. et al., Curr. Opin. Microbiol. doi:10.1016/j.mib.2017.05.014 (2017)). However, their development is limited by the lack of methods for cost-effective and rational design (Mulder K. C. L. et al., Curr. Protein Pept. Sci. 14, 556-567 (2013); Bradshaw J. P. BioDrugs. 17, 233-240 (2003); da Costa J. P. et al., Appl. Microbiol. Biotechnol. 99, 2023-2040 (2015); Fjell C. D. et al., Nat. Rev. Drug Discov. 11, (2011)). Although some alternative methods to overcome these limitations have been proposed (Li Y. Protein Expr. Purif. 80, 260-267 (2011); Ong Z. Y. et al., Adv. Drug Deliv. Rev. 78, 28-45 (2014); Zhao C. X. et al., Biotechnol. Bioeng. 112, 957-964 (2015)), the SAR of these agents is far from understood, which would provide a more substantial basis for their rational design and accelerate their translation to the clinic.
Here, a systematic SAR design approach is described aimed at revealing the sequence requirements for antimicrobial activity of a natural wasp venom AMP (Souza B. M. et al., Peptides 26, 2157-2164 (2005)) and several of its derivatives. Through single-residue substitutions guided by identified physicochemical activity determinants, peptide antibiotics were generated with anti-infective potential in a mouse model.
Pol-CP—NH2 is a chemotactic peptide from the venom of a tropical species of wasp that presents 12 residues typical of peptides found in these wasp species (Souza B. M. et al., Peptides 26, 2157-2164 (2005)). Wasp venom peptides usually present characteristic motifs, such as a Phe-Leu-Pro tripeptide at the amino terminal side, which are thought to be responsible for their mechanism of action. Pol-CP—NH2, however, lacks these specific sequence patterns, which may explain its decreased antimicrobial activity compared to other wasp venom peptides such as mastoparan and VesCP (Souza B. M. et al., Peptides 26, 2157-2164 (2005); Nagashima K. et al., Biochem. Biophys. Res. Commun. 168, 844-849 (1990)). Also unlike other wasp venom peptides, Pol-CP—NH2 lacks a central cationic Lys residue in its seventh position (Nagashima K. et al., Biochem. Biophys. Res. Commun. 168, 844-849 (1990)). Pol-CP—NH2 does contain a Lys residue in its tenth position, like its analog protonectin. The main structural difference between protonectin and Pol-CP—NH2 is the replacement of the eleventh residue in protonectin (Gly) by a Ser in the Pol-CP—NH2 sequence. The differences between Pol-CP—NH2 and other mastoparan-like peptides does not prevent it from presenting chemotactic activity. Pol-CP—NH2 was described as cause of mast cell degranulation activity reduction, mast cell lysis, besides of inducing chemotaxis of polymorphonucleated leukocytes, characteristics usually observed for wasp venom mastoparan-like peptides (Nagashima K. et al., Biochem. Biophys. Res. Commun. 168, 844-849 (1990)).
MIC (
To test the importance of the hydrophilic residues and increased charge in structure-function, synthetic analogs were engineered. Two of these ([Lys]4-Pol-CP—NH2 and [Lys]'-Pol-CP—NH2), which had insertions in the hydrophilic face at positions that would keep the hydrophobicity and hydrophobic moment within the optimal range (
The impact of the introduction of charge via the insertion of Lys residues in positions 4, 5, and 7 was also predicted in the initial experiments (
To analyze the combined effect of charge and the importance of the residues' side chains on the hydrophobic face, other analogs with double ([Leu]5-[Lys]9-Pol-CP—NH2) and triple substitutions ([Glu]3-[Lys]5-[Glu]12-Pol-CP—NH2) were synthesized based on two-dimensional helical wheels (
Results obtained with the newly designed analogs (
A critical design property of AMPs is ensuring their specificity towards microorganisms, while minimizing unwanted toxicity against human cells. To check the toxicity of the second generation of Pol-CP—NH2 derivatives, assays were performed using red blood cells either untreated or exposed to peptides (0-100 mol L−1—
The presence of charged residues on cationic amphipathic AMPs usually correlates with susceptibility to degradation by proteases. Being unstructured in water or saline media, these AMPs are easily cleaved by peptidases. The stability of Pol-CP—NH2 and analogs in fetal bovine serum wash checked for six hours and a small difference was observed in their resistance to degradation (
Among the microorganisms studied, P. aeruginosa is a pathogenic Gram-negative bacterium responsible for pneumonia (El Solh A. A. et al., Am. J. Respir. Crit. Care Med. 178, 513-519 (2008)) and for infections of the urinary tract (Newman J. W. et al., FEMS Microbiol. Lett. 364, fnx124-fnx124 (2017)), gastrointestinal tissue (Yeung C. K. and Lee K. H. J. Paediatr. Child Health 34, 584-587 (1998)), skin and soft tissues (Nagoba B. et al., Wound Med. 19, 5-9 (2017); Dryden M. S. J. Antimicrob. Chemother. 65, iii35-iii44 (2010); Buivydas A. et al., FEMS Microbiol. Lett. 343, 183-189 (2013)) and is very common in patients with cystic fibrosis (Stefani S. et al., Int. J. Med. Microbiol. 307, 353-362 (2017)). Like other bacteria, P. aeruginosa is becoming resistant to common antibiotics (Stefani S. et al., Int. J. Med. Microbiol. 307, 353-362 (2017)), and AMPs have been proposed as an alternative treatment to combat such infections (Chen C. et al., Sci. Rep. 7, 8548 (2017)).
The skin infection mouse model used here involved inducing a P. aeruginosa abscess and treating mice with a single dose of the selected peptides at low concentrations (4 mol L−1) that did not induce hemolysis (
To show the suitability of the lead peptide [Lys]7-Pol-CP—NH2 as a novel peptide antibiotic, its anti-infective activity was tested against P. aeruginosa using the mouse model (
Disclosed herein is a physicochemical feature-guided design of antimicrobial peptides that is a useful tool for identifying functional determinants and designing novel synthetic peptide antibiotics. Using such an approach (Ala-scan and residue probability in determined positions), a naturally occurring AMP has been converted from an AMP with lower activity against Gram-negative bacteria (Souza B. M. et al., Peptides 26, 2157-2164 (2005)), into potent variants capable of killing bacteria at nanomolar doses and displaying anti-infective activity in an animal models. This study is an example of how to design small cationic amphitathic peptides to optimize biological activities and selectivity. The principles and approaches exploited here can be applied to other structure-activity studies in order to rationalize and better understand the role of physicochemical features and which approaches fit better to each family of peptides.
Homology-Based Protein Structure Models. Macromolecular Crystallography, Part D 374,461-491 (Academic Press, 2003).
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are cjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. A
sed herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.
This application claims priority under 35 U.S.C. § 119(e) to U.S. patent application No. 62/734,298, filed Sep. 21, 2018, the entire contents of which are incorporated herein by reference.
This invention was made with Government support under Grant No. HDTRA1-15-1-0050 awarded by the Defense Threat Reduction Agency. The Government has certain rights in the invention
Filing Document | Filing Date | Country | Kind |
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PCT/US2019/051925 | 9/19/2019 | WO | 00 |
Number | Date | Country | |
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62734298 | Sep 2018 | US |