Patent Application
20250127167
The invention generally contemplates peptides and uses thereof as antimicrobial agents.
Infectious diseases severely affect all aspects of human life worldwide and have become a serious public health and economic concern. Nearly one-third of these infections are ascribed to viruses. To date, more than 200 infectious diseases have been discovered; some of them such as influenza A virus subtype H1N1, and H5N1, Ebola, human immunodeficiency viruses (HIV), severe acute respiratory syndrome (SARS), and novel coronavirus disease (COVID-19) lead to high morbidity and mortality levels. In particular, the extraordinary epidemic outbreak of the COVID-19 global pandemic created dramatic challenges to human health and well-being; it adversely affected the world economy and threatened the welfare of populations. The COVID-19 pandemic “wake-up call” has motivated researchers to develop novel strategies and methods to overcoming the spread of viruses. Coronavirus can be easily transmitted by touching surfaces contaminated by it and then touching the face (e.g., the eyes, nose, and mouth), which is considered to be one of the major spread routes.
Moreover, unfortunately, most viruses can survive on surfaces composed of metals, oxides, and polymers for a long time (from tens of hours to several days). For example, it has been reported that the COVID-19 viral pathogens can attach to surfaces and survive for 4 days on glass and ˜7 days on stainless steel and polymeric surfaces. Although some viruses (e.g., HIV) cannot spread outside the body, others, respiratory infection viruses, can easily attach to the surface and remain adherent, which increases the risk of infection. Although direct physical or chemical sanitization by spraying or wiping with disinfectants (such as hydrogen peroxide or rubbing alcohol) is effective to terminate the fomite transmissions through surface touching, the effect of a virus-free environment is not permanent. A better, more effective strategy is to provide the surface with an antiviral activity that will eliminate the viruses quickly after their attachment. Therefore, there is an urgent need to design and develop new, innovative antiviral surfaces and coatings that could be widely applied to various substrates to lower the risk of viral transmission and harm to public health.
Several reports show that some natural extracts (e.g., phenolic compounds, essential oils, and polysaccharides) can be incorporated into coatings to provide antiviral properties. For example, tea tree and eucalyptus oil coatings exhibited good antiviral activity against the influenza virus. However, integrating and applying these oils as effective coatings are somewhat challenging. Another proposed strategy is to incorporate metal nanomaterials such as copper, silver, and zinc, which act as antiviral agents. Inspired by the idea of using nanomaterial-based antiviral agents, researchers have also developed polymeric and organic antiviral coatings. For example, polyethyleneimine-coated glass slides were shown to decrease bacteriophage PRD1 virus titers compared with bare glass. However, the toxicity of both organic and inorganic nanoparticles may cause health and environmental issues. To overcome this problem, researchers have been searching for environmentally friendly and low-toxicity antiviral coatings.
Peptides have also been suggested as antiviral compounds. A 20-amino acid peptide derived from a signal sequence of fibroblast exhibited a broad-spectrum antiviral activity against influenza viruses including the H5N1 subtype. They suggested that the peptide is attached to a cellular receptor and that this mechanism prevents viral infection. In addition, several antimicrobial peptides (AMPs) have been tested as antiviral agents against SARS-CoV-2 and other RNA-based viruses due to their low toxicity. Although significant efforts have been made to develop antiviral peptides as therapeutic agents against viral infections, it would be beneficial to utilize them as antiviral coatings that block the contact transmission of the virus, especially respiratory tract viruses.
Self-assembled peptides can serve as good candidates for fabricating functional coatings. The inventors of the present technology have previously shown that the tripeptide DOPA-Phe(4F)-Phe(4F)—OMe can self-assemble (via the DOPA entity that has “sticky” properties) into a coating that prevents the adhesion of proteins and bacteria on surfaces via the F moiety that has antiadherence characteristics. In addition, they have recently reported that this peptide self-assembles in an aqueous solution into spherical particles. Despite its antuadherence properties, F-based peptides are not antimicrobial [1].
The inventors of the technology disclosed herein have now demonstrated that peptides that lack the antiadherence fluoro atom (F moiety), such as DOPA-Phe(4Br)-Phe(4Br)—OMe and DOPA-Phe-Phe-OMe exhibit antimicrobial and in particular antiviral properties, both in solution and as solid films on various surfaces. As demonstrated, these peptides reduce the number of T4 bacteriophage and canine coronavirus cases by more than 99.9%, opening the door for extensive full spectrum applications in humans, animals and in agricultural uses.
The elimination of an antifouling moiety, such as an F moiety, from short and even ultrashort peptides, has unexpectedly demonstrated increased antimicrobial properties. This trend is highly surprising particularly in view of the fact that the antimicrobial properties were observed also in cases where the peptide did not form into solid films on a surface. Solutions of the F-less peptides were found highly antimicrobial, suggesting that film forming properties are not essential for achieving effective and prolonged antimicrobial properties.
In a first aspect of the invention, there is provided an antimicrobial formulation comprising at least one antimicrobial peptide of structure (I): DOPA-X, wherein X is an amino acid or a peptide comprising between 2 and 4 amino acids, wherein the peptide of structure (I) is free of F atoms or free of antifouling moieties or atoms.
The antimicrobial peptide of structure (I) is a short or an ultrashort peptide comprising overall between 2 and 5 amino acids, one of which being DOPA, as defined herein, and the other being selected amongst aromatic amino acids.
In some embodiments, the antimicrobial peptide is a short peptide of structure (I), wherein X comprises 2 or 3 or 4 amino acids. In some embodiments, the antimicrobial peptide is an ultrashort peptide of the structure (I), wherein X is a single amino acid.
In some embodiments, the antimicrobial peptide of structure (I) is of a structure (II): DOPA-(AA)n-M, wherein
In some embodiments, the peptide of structure (II) is DOPA-(AA)n-O-M (being an ester or an equivalent form of the peptide), wherein each of DOPA, AA, n, O and M is as defined herein.
In other words, a peptide of structure (II) may be generally depicted to be of the short representation DOPA-( . . . )—C(═O)—OM or DOPA-( . . . )—C(═O)—N— . . .
In some embodiments, the peptide of structure (II) is DOPA-(AA)n-NH2, DOPA-(AA)n-NHR1, DOPA-(AA)n-NHR1R2+, or DOPA-(AA)n-NR1R2R3+ (each of which being an amide form of the peptide), wherein each of R1, R2 and R3, independently of the other is —H or a —C1-C5alkyl, and wherein the nitrogen atom is positively charged, the peptide is associated with at least one counter ion. The counter ion may be any single atom anion or an anionic group of atoms. Such counter ions may be a halide (a chloride, a bromide, an iodide), a sulfate, a pyrosulfate, a bisulfate, a sulfite, a bisulfite, a nitrate, a phosphate, a monohydrogen phosphate, a dihydrogen phosphate, a metaphosphate, a pyrophosphate, an acetate, a propionate, a caprylate, an isobutyrate, an oxalate, a malonate, a succinate, a suberate, a sebacate, a fumarate, a maleate, a mandelate, a benzoate, a chlorobenzoate, a methylbenzoate, a dinitrobenzoate, a phthalate, a benzenesulfonate, a toluenesulfonate, a phenylacetate, a citrate, a lactate, a maleate, a tartrate, a methanesulfonate and others.
In some embodiments, each of the amino acids designated AA is an aromatic amino acid. In some embodiments, at least one of the amino acids designated AA is an aromatic amino acid. In some embodiments, at least one of the amino acids designated AA is a brominated or a chlorinated aromatic amino acid.
In some embodiments, the aromatic amino acid is selected from phenylalanine, tryptophan and tyrosine. In some embodiments, the aromatic amino acid is phenylalanine or a derivative thereof.
In some embodiments, the phenylalanine derivatives is 4-methoxy-phenylalanine, 4-carbamimidoyl-1-phenylalanine, 4-chloro-phenylalanine, 3-cyano-phenylalanine, 4-bromo-phenylalanine, 4-cyano-phenylalanine, 4-hydroxymethyl-phenylalanine, 4-methyl-phenylalanine, 1-naphthyl-alanine, 3-(9-anthryl)-alanine, 3-methyl-phenylalanine, m-amidinophenyl-3-alanine, phenylserine, benzylcysteine, 4,4-biphenylalanine, 2-cyano-phenylalanine, 2,4-dichloro-phenylalanine, 3,4-dichloro-phenylalanine, 2-chloro-penylalanine, 3,4-dihydroxy-phenylalanine, 3,5-dibromotyrosine, 3,3-diphenylalanine, 3-ethyl-phenylalanine, 3-chloro-phenylalanine, 3-chloro-phenylalanine, 4-amino-L-phenylalanine, homophenylalanine, 3-(8-hydroxyquinolin-3-yl)-1-alanine, 3-iodo-tyrosine, kynurenine, 3,4-dimethyl-phenylalanine, 2-methyl-phenylalanine, m-tyrosine, 2-naphthyl-alanine, 5-hydroxy-1-naphthalene, 6-hydroxy-2-naphthalene, meta-nitro-tyrosine, (beta)-beta-hydroxy-1-tyrosine, (beta)-3-chloro-beta-hydroxy-1-tyrosine, o-tyrosine, 4-benzoyl-phenylalanine, 3-(2-pyridyl)-alanine, 3-(3-pyridyl)-alanine, 3-(4-pyridyl)-alanine, 3-(2-quinolyl)-alanine, 3-(3-quinolyl)-alanine, 3-(4-quinolyl)-alanine, 3-(5-quinolyl)-alanine, 3-(6-quinolyl)-alanine, 3-(2-quinoxalyl)-alanine, styrylalanine, 4-iodo-phenylalanine, 4-nitro-phenylalanine, phosphotyrosine, 4-tert-butyl-phenylalanine, 3-amino-L-tyrosine, 3,5-diiodotyrosine, 3-amino-6-hydroxy-tyrosine, and/or tyrosine.
In some embodiments, the aromatic amino acid is brominated, e.g., comprises a Br atom, or chlorinated, e.g., comprises a Cl atom, on the aryl functionality (phenyl), at any position of the aromatic ring. In some embodiments, the Br or Cl atom is positioned at the or to (2Br or 2Cl), meta (3Br or 3Cl) or para (4Br or 4Cl) position. In some embodiments, the brominated or chlorinated aromatic amino acid is a brominated or chlorinated phenyl alanine (4Br or 4Cl).
In some embodiments, the aromatic acid is phenyl alanine (Phe) or a brominated or chlorinated phenylalanine (Phe(Br) or Phe(Cl), respectively). In some embodiments, the aromatic amino acid is a brominated phenyl alanine Phe(4Br).
In some embodiments, in a peptide of structure (II), n is 1 or 2. In some embodiments, in a peptide of structure (II), wherein n is 1 or 2, one or both of the amino acids is an aromatic amino acid; or one or both of the amino acids is a brominated amino acid.
In some embodiments, the antimicrobial peptide used according to the invention is selected from:
Formulations of the invention may be designated antimicrobial formulations, namely having themselves antimicrobial properties, or as formulations for forming antimicrobial films on a surface region, or as formulations for preventing attack or damage or degradation or decomposition or poisoning due to presence of a microbial infection source, or as formulations for forming protective antimicrobial films or coats, or as formulations for agricultural uses for applications on live plants, on fruits and vegetables or seeds.
In some embodiments, formulations of the invention may be formed as agricultural formulations for decreasing microbial load in a medium which may be a liquid medium or a surface. The decrease in the microbial load may be by preventing microbial infection, propagation, attachment or spreading; by eradicating (decrease number) of microbial cells or virions in a target (surface, bulk solution), after they have already been established; or by repelling microbial settling or attachment or assembly on a surface of an agricultural product such as live plants, pre-harvested or post-harvested fruits, vegetables, flowers and seeds, for improving their growth, storage, handling, safety, effectiveness and for preventing spoilage and production of microorganism-derived undesirable by-products, such as carbon dioxide, methane, nitrogenous compounds, butyric acid, propionic acid, lactic acid, formic acid, sulfur compounds, and other gases and acids that can have a detrimental effect. In some embodiments, the formulation of the invention prevents propagation and spreading of the microorganism by prevention of assembly and production of new microbial cells or viral capsids.
In some embodiments, formulations or peptides of the invention may be formulated or used as disinfectant compositions or as preservatives. When used as preservatives, the peptides can be incorporated into any suitable product, such as a paint, a latex emulsion, a polymer emulsion, an adhesive, a sealant, a caulk, a mineral or pigment slurry, a printing ink, a pesticide formulation, a household product, a personal care product, a hygiene product, a metal working fluid, a pharmaceutical, a foodstuff, a food additive, any packaging material and the like.
Personal care products that may contain the peptide(s) or topical pharmaceutical formulations which may include an effective amount of a peptide according to the invention may include an emulsion, a cream, a toner, an essence, a pack, a gel, a powder, a makeup base, a foundation, a lotion, an ointment, a patch, a cosmetic solution, a cleansing foam, a cleansing cream, a cleansing water, a body lotion, a body cream, a body oil, a body essence, a shampoo, a rinse, a body cleanser, a soap, a hair dye, a spray, etc.
In some embodiments, formulations or peptides of the invention may be formed into films or coated on a surface region of an object, such as aesthetic objects, cosmetic objects, medical devices, medical surfaces, etc. The antimicrobial coating formed by peptides or formulations of the invention may be for use as a long-term coating, for example, more than 1 month, on a surface of consumer goods containers, means of transport, furnishing objects, common spaces, equipment, clothing, surfaces of medical devices (prostheses, catheters, bandages, and others), or any other surface which favors proliferation of bacterial colonies and the adhesion of viral particles.
Depending on the type of formulation and the intended use, the formulation may comprise one or more active or inactive (or inert) additives or materials. Formulations of the invention may comprise a carrier such as water or other aqueous media, stabilizers, antioxidizing agents, salts, desiccants, defoliants, surfactants, coloring agents, emulsifiers, dispersants, metals such as copper, essential oils, drugs as well as active agents which may be selected and tailored for a specific use.
In some embodiments, formulations of the invention are aqueous formulations, optionally comprising an alcohol.
In some embodiments, formulations of the invention are dispersions, suspensions emulsions, optionally comprising water.
Formulations of the invention may be formed into any type of liquid formulation or a solid formulation, e.g., a solid formulation which can be redispersed or reconstituted in a fluid. The formulations may be formed into aerosol formulations, liquid formulations, spray formulations, dust formulations, dry flowable formulations, granulated formulations, wettable formulations, brushing formulations, polymeric formulations and others.
In some embodiments, the peptide utilized in formulations of the invention is provided in a soluble, dispersed or suspended form. The peptides can also be provided as in dispersible solid forms capable of redissolution or redispersion in a liquid medium, e.g., an aqueous medium.
In some embodiments, the peptide utilized in formulations of the invention is provided in a particulate form or an encapsulated form or present or held within a solid matrix which may be a porous matrix, a soluble matrix, a metallic matrix, a polymeric matrix or any matrix known in the art. In some embodiments, the peptide is encapsulated or held within a solid matrix such as capsules or porous solid materials and may be used as such, wherein optionally the peptide is contained to leech out from the capsule or solid matrix over a period of time, and optionally at a predetermined rate. The delivery profile may be tailored by means known in the art.
The amount of the peptide in a formulation according to the invention may vary based, inter alia, on the type of peptide(s) used, the intended application, the desired effect, etc. Generally speaking, the peptide may be present in an amount ranging between 0.01 and 20 wt %, or between 0.5 and 10 wt %, or between 1 and 5 wt %.
As states herein, in an antimicrobial formulation or combination comprising at least one antimicrobial peptide of the structure (I), as defined, the peptide of structure (I) is free of F atoms or free of any antifouling moiety or atom. In other words, the peptide of structure (I) does not have any group that comprises an F atom, nor any group that can be defined as antifouling or which is defined as an antiadherence group.
The antifouling groups excluded from peptides of structure (I) are those capable, or known from the scientific literature to prevent and control fouling of a surface of a solid composition or an object by minimizing, diminishing, or preventing adhesion of bacteria, viruses, and/or fungi. Examples of such groups include F atoms or groups containing one or more F atoms, each of which being individually excluded from peptides of structure (I), as disclosed herein.
Unlike antifouling peptides, which work in a “Teflon-like” mechanism by preventing adhesion due to the presence of F moieties on the peptide, peptides of the invention exhibit antimicrobial properties which are unique and evident not only when the peptides are formed into a film on a surface region of an object, but more interestingly in solution. When peptides disclosed herein are added into a medium, be it a liquid medium or a solid medium, the peptides are capable of rendering the medium microbe-free by directly interacting with microbes and especially the membranes of the microbes that may be present in the media. Without wishing to rely on mechanistic or theoretical discussion, it is believed that the peptides interact with the microbes to interfere with their ability to assemble and produce transmittable bodies such as virions or new cells. It is further believed that the interaction involves peptide penetration through the microorganism's membrane. Thus, peptides used according to the invention induce not only protection against microorganisms but also eradicate microorganisms present, providing both prevention and eradication (or treatment) modalities also in a liquid medium. Peptides used according to the invention may therefore be considered antibacterial, antifungal, antimycotic, antiparasitic, antiprotozoal, antiviral, antiinfectious, antiinfective and/or germicidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal, protozoacidal, or protozoicidal.
While antifouling materials prevent the adhesion of microorganisms to the surface, compounds of the invention kill, disassemble or prevent new assembly of microorganisms and prevent their proliferations, rendering them ineffective. While antifouling materials may be tested as effective when assembled on a surface of an object, the antimicrobial peptides of the invention are effective in solution or in any other medium, thus presenting a more effective and versatile platform for achieving microbe free media.
In some embodiments, peptides used according to the invention exhibit antiviral, antibacterial and antifungal properties.
Peptides used according to the invention enable inter alia eliminating or decreasing proliferation of existing microorganisms in a target medium or surface and prevention of biofilm generation, formation or growth,
Thus, the term “antimicrobial” encompasses prevention and/or retardation of growth, and/or prevention of accumulation of microorganisms, and or decrease in the number of viable viruses and infective virons, bacteria, undulating bacteria, spirochetes, spores, spore-forming organisms, gram-negative organisms, gram-positive organisms, yeasts, fungi, molds, aerobic organisms, anaerobic organisms and mycobacteria.
Non-limiting examples of microbial organisms that can be controlled using formulations of the invention include
In some embodiments, formulations of the invention are tailored for use as antiviral formulations.
In some embodiments, the antiviral formulations are effective against bacteriophages and viral infectors both in human and non-human animals.
In some embodiments, the viral infector is a human or a canine coronavirus.
In some embodiments, the antimicrobial formulations of the invention are further used against vegetative or dormant forms of bacteria and fungi, such as spores wherein their growth cycle may be controlled using formulations and methods disclosed herein.
The invention further provides use of at least one antimicrobial peptide of structure (I) or (II), as defined herein, as an antimicrobial agent or as an agent capable of rendering antimicrobial properties to an object, to a formulation or to a combination of materials.
The invention further provides use of at least one antimicrobial peptide of structure (I) or (II), as defined herein, in a method of eradicating or reducing microbial population in a solid or liquid medium.
The invention further provides use of at least one antimicrobial peptide of structure (I) or (II), as defined herein, in a method of preparing an antimicrobial formulation, object or surface.
The invention further provides a film comprising a peptide of structure (I) or (II), or a film formed of a formulation comprising a peptide of structure (I) or (II).
Further provided are particles formed of a peptide of structure (I) or (II), wherein said particles are optionally in a form of capsules. The particles may be spherical in shape and may be characterized by surface pores forming an internal or surface volumes or cavities which may or may not contain a component of the formulation in which the particles are formed or contained.
The invention further provides a method of eradicating or reducing a population of microorganism in a solid or liquid medium, the method comprises contacting or adding or treating or allowing interaction of said medium with a peptide of structure (I) or (II) or a formulation comprising same.
As used herein, the expression “eradicating or reducing population” refers to the ability of peptides disclosed herein to reduce a population of or propagation of or to increase ineffectiveness of viruses including infective virons, bacteria, undulating bacteria, spirochetes, spores, spore-forming organisms, gram-negative organisms, gram-positive organisms, yeasts, fungi, molds, aerobic organisms, anaerobic organisms and/or mycobacteria by 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more. In some embodiments, the population is eradicated, namely at least 95, 96, 97, 98 or 99% or 100% of the population is rendered non-viable, non-propagating or non-infective or dead.
The invention further provides a method of inducing or endowing antimicrobial properties to a surface region of an object, the method comprises contacting said surface region with a peptide of structure (I) or (II) or a formulation comprising same and optionally allowing said formulation to form a solid film material of said peptide.
The invention further provides a method of reducing microbial load on or in an object, the method comprising contacting said object with a peptide of structure (I) or (II) or a formulation comprising same and optionally allowing said formulation to form a solid film material of said peptide
The invention further provides a method for protecting live plants, post harvest plants or their parts, fruits, vegetables, seeds, seedings from attack by at least one microbial source or pathogen, the method comprising contacting said live plants, post harvest plants or their parts, fruits, vegetables, seeds, seedings with a peptide of structure (I) or (II) or a formulation comprising same and optionally allowing said formulation to form a solid film material of said peptide.
Further provided is a method of protecting plants including fruits or vegetables, pre- or post-harvesting from attack by a microbial source or a pathogen, the method comprising contacting said fruits or vegetables with a peptide of structure (I) or (II) or a formulation comprising same and optionally allowing said formulation to form a solid film material of said peptide.
The invention further provides a method of protecting seeds against microbial attack by a microbial source or a pathogen, the method comprising contacting said fruits or vegetables with a peptide of structure (I) or (II) or a formulation comprising same and optionally allowing said formulation to form a solid film material of said peptide.
In some embodiments, the contacting may be achievable by any known application method, including spraying, brushing, washing, coating, printing, immersing, dipping or provided via irrigation or sprinkling etc.
Further provided is a kit comprising a formulation of the invention in a mixed or pre-mixed form and instructions of use.
In some embodiments, the kit comprises an amount of a peptide as disclosed herein and a liquid carrier suitable for making the formulation.
In some embodiments, the kit comprises an amount of a peptide as disclosed herein dissolved, suspended or dispersed in liquid carrier.
The invention thus further provides:
An antimicrobial formulation comprising at least one antimicrobial peptide of structure (I): DOPA-X, wherein X is an amino acid or a peptide comprising between 2 and 4 amino acids, wherein the peptide of structure (I) is free of F atoms or free of antifouling moieties or atoms.
In some configurations of the invention, the peptide of structure (I) is a peptide comprising between 2 and 5 amino acids, one of which being the DOPA, and the other amino acids being selected amongst aromatic amino acids.
In some configurations of the invention, the peptide is a peptide of structure (I), wherein X comprises 2 or 3 or 4 amino acids.
In some configurations of the invention, X is a single amino acid.
In some configurations of the invention, the peptide of structure (I) is of a structure (II): DOPA-(AA)n-M, wherein
In some configurations of the invention, the hydroxylated DOPA is hydroxy-DOPA, dihydroxy-DOPA or trihydroxy-DOPA.
In some configurations of the invention, the peptide of structure (II) is DOPA-(AA)n-O-M, wherein each of DOPA, AA, n, O and M is as defined herein.
In some configurations of the invention, the peptide of structure (II) is DOPA-(AA)n-NH2, DOPA-(AA)n-NHR1, DOPA-(AA)n-NHR1R2+, or DOPA-(AA)n-NR1R2R3+, wherein each of R1, R2 and R3, independently of the other is —H or a —C1-C5alkyl, and when the nitrogen atom is positively charged, the peptide is associated with at least one counter ion.
In some configurations of the invention, each of the amino acids designated AA is an aromatic amino acid.
In some configurations of the invention, at least one of the amino acids designated AA is an aromatic amino acid.
In some configurations of the invention, at least one of the amino acids designated AA is a brominated or a chlorinated aromatic amino acid.
In some configurations of the invention, the aromatic amino acid is selected from phenylalanine, tryptophan and tyrosine.
In some configurations of the invention, the aromatic amino acid is phenylalanine or a derivative thereof.
In some configurations of the invention, the phenylalanine derivatives is 4-methoxy-phenylalanine, 4-carbamimidoyl-1-phenylalanine, 4-chloro-phenylalanine, 3-cyano-phenylalanine, 4-bromo-phenylalanine, 4-cyano-phenylalanine, 4-hydroxymethyl-phenylalanine, 4-methyl-phenylalanine, 1-naphthyl-alanine, 3-(9-anthryl)-alanine, 3-methyl-phenylalanine, m-amidinophenyl-3-alanine, phenylserine, benzylcysteine, 4,4-biphenylalanine, 2-cyano-phenylalanine, 2,4-dichloro-phenylalanine, 3,4-dichloro-phenylalanine, 2-chloro-penylalanine, 3,4-dihydroxy-phenylalanine, 3,5-dibromotyrosine, 3,3-diphenylalanine, 3-ethyl-phenylalanine, 3,4-difluoro-phenylalanine, 3-chloro-phenylalanine, 3-chloro-phenylalanine, 2-fluoro-phenylalanine, 4-amino-L-phenylalanine, homophenylalanine, 3-(8-hydroxyquinolin-3-yl)-1-alanine, 3-iodo-tyrosine, kynurenine, 3,4-dimethyl-phenylalanine, 2-methyl-phenylalanine, m-tyrosine, 2-naphthyl-alanine, 5-hydroxy-1-naphthalene, 6-hydroxy-2-naphthalene, meta-nitro-tyrosine, (beta)-beta-hydroxy-1-tyrosine, (beta)-3-chloro-beta-hydroxy-1-tyrosine, o-tyrosine, 4-benzoyl-phenylalanine, 3-(2-pyridyl)-alanine, 3-(3-pyridyl)-alanine, 3-(4-pyridyl)-alanine, 3-(2-quinolyl)-alanine, 3-(3-quinolyl)-alanine, 3-(4-quinolyl)-alanine, 3-(5-quinolyl)-alanine, 3-(6-quinolyl)-alanine, 3-(2-quinoxalyl)-alanine, styrylalanine, 4-iodo-phenylalanine, 4-nitro-phenylalanine, phosphotyrosine, 4-tert-butyl-phenylalanine, 2-(trifluoromethyl)-phenylalanine, 3-amino-L-tyrosine, 3,5-diiodotyrosine, 3-amino-6-hydroxy-tyrosine, tyrosine.
In some configurations of the invention, the aromatic amino acid is brominated or chlorinated on the aryl functionality, at any position of the aromatic ring.
In some configurations of the invention, the brominated or chlorinated aromatic amino acid is a brominated or chlorinated phenyl alanine (4Br or 4Cl).
In some configurations of the invention, in a peptide of structure (II), n is 1 or 2.
In some configurations of the invention, when n is 1 or 2, one or both of the amino acids is an aromatic amino acid; or one or both of the amino acids is a brominated amino acid.
In some configurations of the invention, the peptide is selected from:
In some configurations of the invention, the formulation is in a form of a suspension, a dispersion or an emulsion comprising a liquid medium and the peptide.
In some configurations of the invention, the peptide is provided soluble in a liquid medium.
In some configurations of the invention, the formulation comprises a liquid medium and the peptide, the formulation being for forming antimicrobial films on a surface region, or for preventing attack or damage or degradation or decomposition or poisoning due to presence of a microbial infection source, or for application on live plants, on fruits and vegetables or seeds.
In some configurations of the invention, the formulation is an agricultural formulation for decreasing microbial load in a liquid medium or a surface.
In some configurations of the invention, the formulation is for
In some configurations of the invention, the formulation is for preventing propagation or spreading of microorganism by prevention of assembly and production of new microbial cells.
In some configurations of the invention, the formulation is formulated as a disinfectant composition or as a preservative.
In some configurations of the invention, the formulation is in a form of a paint, a latex emulsion, a polymer emulsion, an adhesive, a sealant, a caulk, a mineral or pigment slurry, a printing ink, a pesticide formulation, a household product, a personal care product, a hygiene product, a metal working fluid, a pharmaceutical, a foodstuff, a food additive, or any packaging material.
In some configurations of the invention, the peptide is provided in a particulate form or an encapsulated form or present or held within a solid matrix.
In some configurations of the invention, the solid matrix is a porous matrix, a soluble matrix, a metallic matrix, or a polymeric matrix.
In some configurations of the invention, the formulation exhibits antibacterial, antifungal, antimycotic, antiparasitic, antiprotozoal, antiviral, antiinfectious, antiinfective and/or germicidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal, protozoacidal, or protozoicidal properties.
In some configurations of the invention, the formulation is an antiviral formulation.
In some configurations of the invention, the formulation is effective against bacteria from the genus Aeromonas, Arcobacter, Bacillus, Brochothrix, Campylobacter, Carnobacterium, Clostridium, Enterobacteriacae, Escherichia, Listeria, Pseudomonas, Salmonella, Serratia, Shigella, Staphylococcus, Vibrio, Yersinia, Erwinia, Pseudomonas pyocyanea, and Corynebacterium xerosis.
In some configurations of the invention, the formulation is effective against fungi selected from Aspergillus flavum and Penicillium chrysogenum; parasites, Balantidium, Cryptosporidium, Cyclospora, Giardia, Isospora, Microsporidia, Trichinella spiralis and Toxoplasma gondii; Fusarium oxysporum, Penicillium italicum, Colletotrichum gloeosporioides, Colletotrichum capsica, and Fusarium solani, Pythium, Pythium sp., and Sclerotium rolfsii.
In some configurations of the invention, the formulation is effective against viruses and infective virons of viruses selected from bacteriophages, coronaviridae/corona-virus, orthomyxoviridae, paramyxoviridae, Coxsackie family of viruses and adenoviridae family, Tobamovirus, and Tomato brown rugose fruit virus.
In some configurations of the invention, the formulation is effective against bacteriophages and viral infectors in human and non-human animals.
In some configurations of the invention, the formulation is an antiviral formulation wherein the viral infector is a human or a canine coronavirus.
Use of at least one antimicrobial peptide of structure (I) or (II), as defined herein, as an antimicrobial agent or as an antimicrobial agent for rendering antimicrobial properties to an object, to a formulation or to a combination of materials.
Use of at least one antimicrobial peptide of structure (I) or (II), as defined herein, in a method of eradicating or reducing microbial population in a solid or liquid medium.
Use of at least one antimicrobial peptide of structure (I) or (II), as defined herein, in a method of preparing an antimicrobial formulation, object or surface.
A film comprising a peptide of structure (I) or (II), as defined herein, or a film formed of a formulation comprising a peptide of structure (I) or (II).
Particles formed of a peptide of structure (I) or (II), as defined herein, wherein said particles are optionally in a form of capsules.
A method of eradicating or reducing a population of microorganism in a solid or liquid medium, the method comprises contacting or adding or treating or allowing interaction of said medium with a peptide of structure (I) or (II) as defined herein, or a formulation comprising same.
A method of inducing or endowing antimicrobial properties to a surface region of an object, the method comprises contacting said surface region with a peptide of structure (I) or (II) as defined herein or a formulation comprising same and optionally allowing said formulation to form a solid film of said peptide.
A method of reducing microbial load on or in an object, the method comprising contacting said object with a peptide of structure (I) or (II) as defined herein or a formulation comprising same and optionally allowing said formulation to form a solid film of said peptide A method or protecting live plants, post-harvest plants, fruits and vegetables, seeds, or seedings from attack by at least one microbial source or pathogen, the method comprising contacting same with a peptide of structure (I) or (II) as defined herein or a formulation comprising the peptide and optionally allowing said formulation to form a solid film of said peptide.
A method of protecting seeds against microbial attack by a microbial source or a pathogen, the method comprising contacting said seeds with a peptide of structure (I) or (II) as defined herein or a formulation comprising same and optionally allowing said formulation to form a solid film of said peptide.
A film or a coat formed of a formulation as defined herein.
In some configurations of the invention, the film is on a surface of an object.
In some configurations of the invention, the film is formed on an object selected from aesthetic objects, cosmetic objects, medical surfaces, consumer goods containers, means of transport, furnishing objects, common spaces, equipment, clothing, surfaces of medical devices, or a surface which favors proliferation of bacterial colonies and the adhesion of viral particles.
An antimicrobial formulation comprising at least one antimicrobial peptide of structure (I): DOPA-X, wherein X is an amino acid or a peptide comprising between 2 and 4 amino acids, wherein the peptide of structure (I) is free of F atoms or free of antifouling moieties or atoms, the formulation being in a form of an aerosol formulation, liquid formulation, spray formulation, dust formulation, dry flowable formulation, granulated formulation, wettable formulation, brushing formulation, or a polymeric formulation.
A solid matrix comprising an antimicrobial peptide of structure (I): DOPA-X, wherein X is an amino acid or a peptide comprising between 2 and 4 amino acids, wherein the peptide of structure (I) is free of F atoms or free of antifouling moieties or atoms.
In some configurations of the invention, the solid matrix is a porous solid material.
An antiviral liquid reagent comprising a peptide of structure (I) or (II) as defined herein, the reagent being for use as a disinfectant.
A disinfectant or a sterilizing agent comprising a liquid medium and a peptide of structure (I) or (II) as defined herein.
An antimicrobial formulation comprising an antimicrobial peptide consisting a peptide of structure (I) or (II) as defined herein.
An antiviral liquid formulation comprising at least one peptide of structure (II): DOPA-(AA)n-M, wherein
In some configurations of the invention, the formulation comprises the peptide comprises two or three amino acids.
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Peptides were purchased from GL Biochem (Shanghai) Ltd. with a purity >95%. L-DOPA, with a purity >98% was purchased from Tokyo Chemical Industry Co., Ltd. Diphenylalanine (H-Phe-Phe-OH), L-phenylalanine (L-Phe) was purchased from Bachem AG (Bubendorf, Switzerland) Co., Ltd. with a purity of 98%. Methanol, sodium dodecyl sulfate (SDS), and ethanol were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Escherichia coli strain B (Migula) Castellani and Chalmers (ATCC 11303) and Escherichia coli bacteriophage T4 (ATCC 113030-B4) bacteria were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Agar and LB broth were purchased from Merck (New Jersey, USA) and Becton Dickinson (New Jersey, USA), respectively. The CRFK (ATCC® CCL-94™) cell line and Canine coronavirus (CCV) (ATCC® VR2068™) were purchased from Biological Industries, Beit-Haemek, Israel. Essential minimum Earl salts medium (EMEM), 200 mM L-Alanyl-L-Glutamine, Penicillin-Streptomycin-Amphotericin (PSA), Trypsin-EDTA, Dulbecco's Phosphate Buffered Saline (DPBS), and Donor Horse Serum (DHS) were purchased from Biological Industries, Beit-Haemek, Israel. Trypsin 1:250 was purchased from Bio-World, Dublin, OH, USA.
The peptide stock solution was prepared by dissolving the lyophilized peptide powder in ethanol. Then, the stock solution was diluted by triple distilled water (TDW) and stirred for 3 h (150 rpm, room temperature). The final peptide concentration was 5.54 mM.
Prior to the coating procedure, glass surfaces (1 cm*1 cm) were washed for 30 min with each of three different solvents: 2% SDS, methanol, and Ethanol. Subsequently, the surfaces were dried by a flow of nitrogen and then left in UV-Ozone for 10 min. The single-layer coating was prepared by drop-casting 100 μL of peptide (10 mg/mL) solution on clean glass and then dried at room temperature. Subsequently, the double-layer coating was prepared by adding 100 μL peptide solution (10 mg/mL) on the dried 1-layer coating and finally dried at room temperature. The triple-layer coating was prepared by drop-casting three times as described before.
Contact angle measurements were examined by using a Theta Lite optical tensiometer (Attension, Finland). Each experimental measurement consisted of three repeats.
The different dried surfaces were coated with gold using a Polaron SC7640 Sputter Coater. SEM images were taken using an extra high-resolution scanning electron microscope, Magellan TM400L, operating at 1 kV. The coverage degree of the surface by the structures was assessed using ImageJ. It was calculated using eq 1.
X-ray photoelectron spectroscopy (XPS) analyses were performed by using an AXIS Ultra X-ray photoelectron spectrometer (Kratos Analytical, Ltd., Manchester, UK). The sample's take-off angle was 90°. The vacuum pressure in the analyzing chamber was maintained at 2×10−9 Torr. High-resolution XPS spectra were collected for F is, O is, C is, and N is peaks with 20 eV pass energy and 0.1 eV step size. Data analyses were carried out using Kratos Vision data reducing processing software (Kratos Analytical, Ltd.) and Casa XPS (Casa Software, Ltd.).
To examine the coating stabilities, peptide coatings were immersed into TDW for 5 min or washed with 2 mL TDW three times and then dried at room temperature. X-ray photoelectron spectroscopy (XPS) analyses were performed for the immersed and washed coatings. Moreover, peptide coatings were wiped with a finger at a load of 1 g and then water contact angle measurements were performed using the same method as described in the Contact angle measurements section.
FT-IR was recorded using a Nicolet 6700 FT-IR spectrometer with a deuterated triglycine sulfate (DTGS) detector (Thermo Fisher Scientific, MA, USA). Peptide solutions were deposited on a CaF2 plate and dried by vacuum. The peptide deposits were resuspended with D2O and subsequently dried, forming thin films. The resuspension procedure was repeated twice to ensure maximal hydrogen-to-deuterium exchange. The measurements were taken using a 4 cm−1 resolution and averaged after 2000 scans. For ATR-FTIR analysis, ATR spectra were recorded using FT-IR (Thermo scientific, Model Nicolet 6700) with GeATR arrangement (Harrick Scientific's VariGATR). For all the surfaces spectra were 3 collected with an applied force of 350 N, at 4 cm−1 resolutions with 3000 scans averaged signal and an incident angle of 65°.
The antiviral activity performance was measured according to work by Kim et al. and Matsumoto et al. with some modification. The peptide coatings were prepared as described before. We used T4 bacteriophage as the virus when we measured the antiviral activity. Bacteriophage suspension was propagated on E. coli (ATCC 11303) according to ISO 18061, which was grown as the host strain. The mixture obtained by adding 100 μL of fresh T4 phage to an overnight culture of E. coli was incubated for 4 h in a 10 mL LB phage at 37° C. After multiplication, the virus was collected by centrifugation at 4000×g for 10 min at room temperature. The concentration of T4 bacteriophage was measured using the soft agar overlay (double-agar layer) plaque assay method. The supernatant containing the bacteriophage culture was filtrated through cellulose acetate filters (Millipore Acrodisc; the pore sizes were 0.45 μm and 0.22 μm) before use (the final concentration of bacteriophage was 109 PFU/mL).
Ten decimal serial dilutions of the virus suspension were prepared by LB phage. The aqueous suspensions for the E. coli and T4 bacteriophage inactivation experiments contained sample surfaces having dimensions of 1*1 cm and T4 phage at 1.0×106 plaque-forming units (PFU)/mL. As a positive control, we also added 16 μL 1.0×106 plaque-forming units (PFU)/mL bacteriophage T4 on bare glass and then treated the surface by a UV irradiation (long wave ultraviolet 365 nm) for 3 h. Next, the phages were incubated under humid conditions at room temperature (25° C.) in a dark room for 24 h. After incubation, the phages were harvested by shaking with 2 mL SCDLP broth for 15 min to stop the incubation. The T4 in SCDLP bacteriophage was diluted with LB phage 10-fold. Subsequently, samples and bacteria were mixed with 0.6% agarose. Then the mixture was spread out on 1.5% LB agar to form a double agar layer. The plate was incubated at 37° C. for 18 h to form the plaques. The antiviral activity was defined and calculated as eq 2 below: The initial virus titer (No) and the virus titer after incubation (N) were calculated by counting the plaque number. For each sample, 9 repeats were performed to assess the antiviral activity.
All the tripeptide powders were firstly dissolved into ethanol and diluted by TDW. The solutions were stirring for 3 h (150 rpm, room temperature) for complete self-assembly. The final concentration is 10 mg/mL. Our previous study showed that the peptide could self-assemble into spheres. As a control, single amino acids (L-DOPA, L-Phenylalanine, and 4-Fluoro-L-phenylalanine) were also used to examine their antiviral activity. To further evaluate the antiviral activity for the peptide assemblies, the antiviral MIC for traditional Ag and Cu nanoparticles were also tested. The stock solutions were two-fold diluted using LB phage to obtain a series of peptide concentrations until the lowest concentration needed was achieved. Next, the 100 μL T4 bacteriophage (105 PFU/mL) and 100 μL diluted peptide solutions were transferred into 800 μL LB phage and then the samples were shaken at 150 rpm at room temperature. After 24 h incubation, the samples were centrifuged at 14000 g for 10 min to precipitate the peptide particles. Supernatants were collected and then 10-fold diluted once. Then, the 20 μL supernatants were mixed with 25 μL bacteria (E. coli. ATCC11303) in 1 mL warm 0.6% agarose. Then the mixture was spread out on 1.5% LB agar to form a double agar layer. The plate was incubated at 37° C. for 18 h to form the plaques. The lowest concentration of peptides that prevented virus growth is defined as the antiviral minimum inhibitory concentration (MIC).
Cell Culture: CRFK cells were propagated in a growth medium containing EMEM with 2 mM L-Alanyl-L-Glutamine, 1% PSA solution, and 10% DHS at 37° C. with 5% CO2.
Virus propagation: CRFK cells were incubated in a 175 cm2 flask until 80-90% confluent was achieved. After the flask was washed with 20 mL of DPBS, 2 mL of CCV solution were added and incubated at 37° C. with 5% CO2. After a 2-hour incubation, EMEM (with 2 mM L-Glutamine and 1% PSA), DHS (final concentration 1%) and 1:250 Trypsin (final concentration 1 μg/mL) were added to the flask to a final volume of 20 mL. After the infected flask was incubated at 37° C. with 5% CO2 for a week, the medium was centrifuged at 1000×g, at 4° C., for 10 min. Next, the supernatant was transferred to a sterile 20 mL Vivaspin tube with a 100,000 MWCO filter and then centrifuged at 2500 g, at 4° C. to concentrate the virus to a final volume of 1-2 mL and finally preserved in liquid nitrogen.
TCID50/mL Measurements: About 10,000 CRFK cells were incubated in each well using 96-well plates until 80-90% confluent was achieved. Then the examined CCV solution was diluted to a 10-fold dilution series. After the flask was washed with 0.2 mL of DPBS, 0.1 mL of the diluted CCV solution was added and incubated at 37° C. with 5% CO2 (5-6 repeats for each dilution). After a 2-h incubation, EMEM (with 2 mM L-Glutamine and 1% PSA), DHS (final concentration 1%) and 1:250 Trypsin (final concentration 1 μg/mL) were added to the well to a final volume of 0.2 mL. The TCID50/mL (tissue culture infectious dose at the 50% endpoint per mL) calculations were performed according to the improved Karber method.
Antiviral activity for peptide coatings: A 16 μL test viral suspension was inoculated onto the coated and uncoated glass surface and incubated for 3 h at room temperature. As a positive control, we added 16 μL viral suspension and incubated the substrate for 3 h under UV irradiation (longwave ultraviolet 365 nm). After the contact time, a 2 mL SCDLP broth was added to remove the viruses from the surface. From this mixture, a 10-fold dilution series was prepared and a TCID50/mL measurement was carried out. Each experiment contained three coated surfaces and three uncoated surfaces, and three experiments were performed.
Antiviral activity for peptide solutions: A 20 μL viral suspension was inoculated into 180 μL peptide solution (10 mg/mL, 1 mg/mL, 0.1 mg/mL, 0.01 mg/mL, and 0 mg/mL) for 3 h at room temperature. From this mixture, a 10-fold dilution series was prepared and a TCID50/mL measurement was carried out. Each sample at different concentrations was performed by three experiments.
To self-assemble the peptides, we first dissolved the peptide in ethanol to a concentration of 100 mg/mL and then diluted them by triple distilled water to a concentration of 10 mg/mL. The peptide solutions were stirred for 3 h at 150 rpm. This process leads to the formation of spherical structures. To evaluate the antiviral activity of the assemblies, we measured the antiviral minimum inhibitory concentration (MIC) against T4 bacteriophage (see method section). The results are shown in Table 1. Peptide assemblies formed showed antiviral activity against T4 bacteriophage. The antiviral MIC were 62 g/mL and 31 g/mL. This indicated that the assemblies formed by the peptide have better antiviral activity against T4 bacteriophage. For comparison, we assessed the antiviral activity of metal nanoparticles and found that the MIC of silver nanoparticles and copper nanoparticles was 100 g/mL and 2500 g/mL (Table 1). This suggests that the peptide assemblies have better antiviral activity than those of traditional nanoparticles. We also determined the antiviral MIC against T4 bacteriophage for amino acids that comprises the peptides: L-DOPA, L-Phenylalanine, or 4-Fluoro-L-phenylalanine. The MIC values indicate that the individual amino acids have no antiviral activity against T4 bacteriophage (Table 1). These findings suggest that the antiviral activity can be attributed to the assemblies formed by the peptide and not by their components.
To generate a coating from these assemblies we simply drop-casted the peptide solution on a glass slide (
To characterize the morphology of the peptide-based coatings, we analyzed the surface using scanning electron microscopy (SEM).
Next, we analyzed the structural properties of the spherical assemblies formed by the peptides using Fourier Transform Infrared spectroscopy (FT-IR). The spectrum for the assemblies formed in solution by the peptide DOPA-Phe(4F)-Phe(4F)—OMe had two distinctive peaks centered around 1606 cm−1 and 1667 cm−1 (
To determine the hydrophobicity of the coatings, we measured the water contact angle of the coated surfaces (
To ensure the modification of the glass surfaces by the peptides, we measured the X-ray photoelectron spectroscopy (XPS). The atomic concentration of carbon, fluorine, and nitrogen increased upon increasing the number of DOPA-Phe(4F)-Phe(4F)—OMe peptide layers (
To investigate the stability of the coating on the surface, we performed XPS analysis for a 3-layer coating for both peptides, after immersing the coating in a water bath containing water (TDW) for 5 min or washing it three times using a pipette filled with TDW. Importantly, the intensity of the fluorine, carbon, and nitrogen signals did not decrease after this treatment (
To determine the antiviral activity of the peptide-based coating, we incubated about 106 T4 bacteriophages on each surface. T4 bacteriophage is a DNA-based virus that infects E. coli and causes them to burst.
Based on these results, we can conclude the DOPA is not essential for antiviral activity; however, it is necessary when the peptide interacts with a glass surface. Several studies showed that phenylalanine and its derivatives have antiviral and antifungal activity. Glycyrrhizic acid conjugates with phenylalanine and has antiviral activity against H1N1. Benzenesulfonamide-containing phenylalanine derivatives inhibit HIV-1 capsid formation. In addition, fluorinated compounds are known as antiviral agents. This includes 3′-fluoropenciclovir analogues, polyfluoroflavones, perfluoroalkyl derivatives of teicoplanin and vancomycin, 6′-fluorinated-aristeromycin analogues, fluorinated TiO2, and other fluorinated molecules. These observations can explain our results with both DOPA-Phe-Phe and DOPA-Phe(4F)-Phe(4F).
It is important to note that the assemblies formed by Phe-Phe (the assemblies were prepared according to our previous study) and deposited on the glass did not exhibit antiviral activity. We assume that this is due to the lack of DOPA in the sequence and consequently, the instability of the peptide assemblies on the surface.
To examine the antiviral activity of the peptide with coronavirus, we used a corona surrogate—canine coronavirus (CCV). This is an enveloped, positive-stranded RNA virus with specific sequence homology with SARS-CoV-2 (36.93% sequence homology to SARS-CoV-2 of spike protein). Briefly, we inoculated CCV with a concentration (log (TCID50/mL)) of 6.79±0.17 on uncoated and coated glass surfaces for 3 hours. The log (TCID50/mL) (
Two dipeptides, DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2, were examined for their antiviral activity in solution and as a coating. The two peptides comprise one 3,4-dihydroxy-L-phenylalanine (DOPA) and one phenylalanine or fluorinated phenylalanine. L-DOPA is the main constituent of mussel adhesive proteins (MAPs) that can adhere to almost any substrate and can function under harsh conditions. Phenylalanine and fluorinated phenylalanine are aromatic residues that can mediate peptide self-assembly through π-π stacking.
To investigate the antiviral activity of DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2 in solution, we performed an antiviral assay to identify the minimal inhibitory concentration (MIC) of each peptide. We first dissolved the peptides in ethanol to a concentration of 100 mg/mL and then diluted the solutions with triple distilled water to several concentrations. Both DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2 exhibited antiviral activity against bacteriophage T4. The antiviral MIC was 125 g/mL and 62.5 μg/mL for DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2, respectively, indicating that DOPA-Phe(4F)—NH2 had better antiviral activity than DOPA-Phe-NH2. This suggests that fluorinated residues can enhance the antiviral activity of the peptide. It follows our previous report on self-assembled tripeptides that have a similar antiviral activity with antiviral MIC of tens of g/mL.25 The importance of phenylalanine and fluorinated phenylalanine in antiviral peptides has been reported before. The improvement in antiviral activity upon the introduction of fluorine atoms is also in accordance with several previous reports that show that incorporating one or several fluorine atoms into an organic molecule can improve the pharmacokinetic and pharmacodynamic properties such as absorption, tissue distribution, secretion, the route and rate of biotransformation, toxicology, bioavailability, metabolic stability, and lipophilicity. Specifically, it was demonstrated that the peptide carbobenzoxy-D-phenylalanine-L-phenylalanine-glycine acts as an inhibitor of membrane fusion. In addition, it was shown that the fluoro-group at the phenyl ring in a triazole-dipeptide hybrid is essential for the antiviral peptide activity. To explore the antiviral mechanism for the dipeptide assemblies, we also evaluated the antiviral activity against bacteriophage T4 for L-DOPA, L-phenylalanine(L-Phe), fluorinated phenylalanine (L-Phe(4F)), diphenylalanine (Phe-Phe), and fluorinated diphenylalanine (Phe(4F)-Phe(4F)—OMe). As shown in Table 2, a single amino acid and the dipeptides Phe-Phe and Phe(4F)-Phe(4F)—OMe did not decrease the viral titer. We included copper nanoparticles (CuNPs) as a positive control in the antiviral MIC experiment. The antiviral MIC for CuNPs is 2500 g/mL much higher than the peptides MIC.
To investigate in what form the peptides exist in the solution at the MIC concentration, a transmission electron microscope (TEM) was used. The TEM analysis for the dipeptide DOPA-Phe-NH2 revealed that the peptide self-assembles into spherical nanoparticles with a size ranging from several nanometers up to 20 nm (
To better understand the mechanism of antiviral activity, we incubated a solution of bacteriophage T4 with the peptide assemblies at the peptide MIC concentration for 24 h and performed a TEM analysis (
To further utilize the self-assembled particles to generate an antiviral coating we used a protocol we reported before. A peptide solution of DOPA-Phe-NH2 or DOPA-Phe(4F)—NH2, at a concentration of 10 mg/mL, was drop-casted three times on a clean glass substrate. This process resulted in transparent surfaces (
We investigated the antiviral activity of the peptide-based coatings against bacteriophage T4. A solution of bacteriophages at a concentration of 1.0×106 plaque-forming units (PFU)/mL was applied to the peptide-based coating and incubated for 24 h. Then, the virus titer was determined by counting the number of plaques. As shown in
The cytotoxicity of the peptides was measured toward both colorectal adenocarcinoma (HT-29) and ovarian carcinoma (A2780) cancer cell lines. The cytotoxicity was tested by using the MTT (3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) assay after incubation of the cells with the peptides for 24 h. Both peptides showed very low cytotoxicity toward the HT-29 cells (
Phe(4F)-Phe(4F)—OMe, DOPA-Phe-NH2, and DOPA-Phe(4F)—NH2 were purchased from GL Biochem (Shanghai) Ltd. with a purity>95%. L-DOPA with a purity >98% was purchased from Tokyo Chemical Industry Co., Ltd. L-Phenylalanine, L-Phe(4F), and Diphenylalanine (H-Phe-Phe-OH) were purchased from Bachem AG (Bubendorf, Switzerland) Co., Ltd. with a purity of 98%. Methanol, sodium dodecyl sulfate (SDS), ethanol, Roswell Park Memorial Institute (RPMI) 1640 medium, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) and Isopropanol were obtained from Sigma Aldrich. were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Escherichia coli strain B (Migula) Castellani and Chalmers (ATCC 11303) and Escherichia coli bacteriophage T4 (ATCC 113030-B4) bacteria were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Agar and LB broth were purchased from Merck (New Jersey, USA) and Becton Dickinson (New Jersey, USA), respectively. Ovarian carcinoma A2780 was purchased from European Collection of Authenticated Cell Cultures and colorectal adenocarcinoma HT-29 was purchased from American Type Culture Collection. Fetal Bovine Serum, 1% Penicillin-Streptomycin, and 1% L-Glutamine, were purchased from Biological Industries (Beit Haemek, Israel).
Preparation of the Peptide Assemblies and Peptide Coatings The dipeptide assemblies were prepared by dissolving the peptide powder in ethanol at 100 mg/mL; then they were diluted using triple distilled water (TDW) according to our previous work.16 The peptide coatings were prepared by drop-casting 3 times with 10 mg/mL peptide assemblies.
A series of two-fold diluted peptide solutions (1000, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 g/mL) was prepared to measure the antiviral MIC. Next, 100 μL T4 bacteriophage (105 PFU/mL) and 100 μL diluted peptide solutions were transferred into 800 μL LB phage; then, the samples were shaken at 150 rpm at room temperature. After 24 h incubation, the samples were centrifuged at 14000×g for 10 min to precipitate the peptide particles. Supernatants were collected and then 10-fold diluted once. Next, the 20 μL supernatants were mixed with 25 μL bacteria (E. coli. ATCC11303) in 1 mL of warm 0.6% agarose. Then, the mixture was spread out on 1.5% LB agar to form a double agar layer. The plate was incubated at 37° C. for 18 h to form plaques. The lowest concentration of peptides that prevented virus growth is defined as the antiviral minimum inhibitory concentration (MIC). L-DOPA, L-phenylalanine, L-Phe(4F), Phe(4F)-Phe(4F), and diphenylalanine were used as controls.
The peptide assemblies at antiviral MIC (125 g/mL and 62.5 g/mL for DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2, respectively) were characterized by using Tecnai 12 TEM 120 kV (Phillips, Eindhoven, the Netherlands). Firstly, a carbon Formvar-coated copper grid was placed on a drop of peptide solution. Then, the samples were negatively stained by adding 5 μL of 2% uranyl acetate for 40 sec and dried in room temperature. The samples were transferred to TEM characterization immediately after preparation.
SEM measurements were performed by an extra high-resolution scanning electron microscope, Magellan TM400L, operating at 1 kV. A solution of peptide assemblies at antiviral MIC (125 g/mL and 62.5 g/mL for DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2, respectively) was drop casted on glass surface and allowed to dry at room temperature. Then, the peptide assemblies and coatings were coated with gold using a Polaron SC7640 sputter coater and then observed.
The peptide assemblies were prepared at antiviral MIC (125 g/mL and 62.5 g/mL for DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2, respectively) and then were drop-casted on the clean glass substrate. All AFM images of the peptide assemblies and coatings were taken in AC mode with a Si3N2 tip with a spring constant of 3 N/m using JPK NanoWizard®.
A Malvern dynamic laser scattering (DLS) instrument (Zetasizer Nano ZSZEN3600) was used to determine the size distribution of the peptide assemblies. The size distribution of peptide assemblies at antiviral MIC (125 g/mL and 62.5 g/mL for DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2, respectively) was performed.
The CD spectra were collected in a J-810 spectropolarimeter (JASCO, Tokyo, Japan), using a 0.1 cm pathlength quartz cuvette for far-UV CD spectroscopy (in the spectral range between 190 and 260 nm with a step width of 0.05 nm) at 20° C. The peptides were dissolved in TDW (0.1 mg/ml) and then filtered by using a 0.22 μm filter. The spectra for each sample spectra were collected three times, averaged, and the background (TDW) was subtracted.
FT-IR was recorded using a Nicolet 6700 FT-IR spectrometer with a deuterated triglycine sulfate (DTGS) detector (Thermo Fisher Scientific, MA, USA) at a 4 cm-1 resolution and averaged after 2000 scans. Peptide solutions were deposited on a CaF2 plate and dried by vacuum. The peptide deposits were resuspended with D2O and subsequently dried, forming thin films. The resuspension procedure was repeated twice to ensure maximal hydrogen-to-deuterium exchange.
The phage stock solution at a concentration of 1·109 PFU/ml was diluted to 6-107 PFU/ml in DDW and added to either the peptide solution or TDW. The final concentration of the peptides DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2 were 25 mg/ml and 12.5 mg/ml, respectively. The solutions were incubated for 24 h at 37° C., 120 rpm.
Then, a 10 μL drop of each sample was added to the grid for 30 sec and the excess was blotted with filter paper. The samples were negatively stained by adding 5 μL of 2% uranyl acetate for 40 sec and the excess was blotted with filter paper.
The samples were analyzed by using Tecnai 12 TEM 120 kV (Phillips, Eindhoven, the Netherlands) equipped with Phurona camera and RADIUS software (Emsis GmbH, Münster, Germany).
ATR-FTIR spectra were collected with an applied force of 350 N, at 4 cm-1 resolution with 3000 scans averaged signal and an incident angle of 65°.
The antiviral activity performance was measured according to our previous work. Briefly, 10 decimal serial dilutions of the virus suspension were prepared by LB phage. The aqueous suspensions for the E. coli and T4 bacteriophage inactivation experiments contained sample surfaces with dimensions of 1*1 cm and T4 phage at 1.0×106 plaque-forming units (PFU)/mL. Next, the phages were incubated under humid conditions at room temperature (25° C.) in a dark room for 24 h. After incubation, the phages were harvested by shaking them with 2 mL SCDLP broth for 15 min to stop the incubation. The T4 in the SCDLP bacteriophage was diluted with LB phage 10-fold. Subsequently, samples and bacteria were mixed with 0.6% agarose. Then, the mixture was spread out on 1.5% LB agar to form a double agar layer. The plate was incubated at 37° C. for 18 h to form the plaques. The antiviral activity was defined and calculated as follows: The initial virus titer (NO) and the virus titer after incubation (N) were calculated by counting the plaque number. For each sample, 9 repeats were performed to assess the antiviral activity.
Colorectal adenocarcinoma (HT-29) and Ovarian carcinoma (A2780) cancer cell lines were cultured as monolayers at 37° C. in a 5% CO2 atmosphere, in RPMI 1640 medium, supplemented with 10% Fetal Bovine Serum, 1% Penicillin-Streptomycin, and 1% L-Glutamine. Cytotoxicity was measured by the previously reported MTT method.36 The cells were seeded in a 96-well plate, at a density of ca. 10000 cells per well, and allowed to attach overnight under the conditions mentioned above. The peptides DOPA-Phe-NH2 and DOPA-Phe(4F)—NH2 were dissolved in ethanol to 291 mM and 276 mM, respectively, and then diluted in TDW to a 2 mM concentration. The samples were then serially diluted to create a concentration gradient, with pure TDW as the control, and added to the cells so that the highest concentration was set to 100 PM. The plate was incubated for 24 hours under the same conditions. MTT, 0.1 mg in 20 μl, was added to each well, followed by an additional three-hour incubation. The medium was removed and 200 μl of isopropanol were added to each well, and the absorbance at 550 nm was measured (Spark 10 M multimode microplate reader spectrophotometer, Tecan Group Ltd. Mannedorf, Switzerland). Cell viability was calculated by comparing the formazan absorbance in the treated wells to the untreated control wells. Each measurement was repeated in three wells per plate, and at least on three different days, to total at least 9 repetitions. The relative IC50 values and the standard error of the means were determined by nonlinear regression of a variable slope (four parameters) model, using the GraphPad Prism 5.0 software.
The potential antiviral activity of DOPA-Phe(Br)—OH against fruit viruses and other microbial sources was also investigated. Various brominated peptides, including DOPA-Phe(Br)—OH and DOPA-Phe(Br)—NH2, were tested and found capable of eradicating fruit and plant viruses.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2022/050994 | 9/14/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63261204 | Sep 2021 | US |
