WO2005123105A1 - Phototoxic compounds - Google Patents

Phototoxic compounds Download PDF

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Publication number
WO2005123105A1
WO2005123105A1 PCT/US2005/020629 US2005020629W WO2005123105A1 WO 2005123105 A1 WO2005123105 A1 WO 2005123105A1 US 2005020629 W US2005020629 W US 2005020629W WO 2005123105 A1 WO2005123105 A1 WO 2005123105A1
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WIPO (PCT)
Prior art keywords
amino acid
peptide
phototoxic
dna
side chain
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PCT/US2005/020629
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French (fr)
Inventor
Shana O. Kelley
Jay Carreon
Lisa Wittenhagen
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The Trustees Of Boston College
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Publication of WO2005123105A1 publication Critical patent/WO2005123105A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT

Definitions

  • the invention relates to a phototoxic compound that includes a photosensitizer conjugated to a peptide or peptoid moiety.
  • the phototoxic compound mediates light-induced cleavage of DNA in a biological system.
  • the invention further relates to the use of the phototoxic compound for inducing cytotoxic activity in a cell and for photodynamic therapy.
  • Photodynamic therapy also referred to as photosensitization-based therapy (PDT), photoradiation therapy, phototherapy or photochemotherapy
  • PDT photosensitization-based therapy
  • Photoradiation therapy is a medical treatment that employs a combination of light and a photosensitizing agent to generate a cytotoxic effect of cancerous or other unwanted tissues or organisms
  • PDT photosensitization-based therapy
  • Photoradiation therapy is a medical treatment that employs a combination of light and a photosensitizing agent to generate a cytotoxic effect of cancerous or other unwanted tissues or organisms
  • Photodynamic therapy consists of introducing a photoactive drug into a body and subsequent illumination of cells by visible or near infrared light. In the presence of oxygen, illumination activates the drug and in turn produces reactive oxygen species which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids, leading to cell destruction.
  • Photodynamic therapy has been effective in treating multiple types of cancer, including cancers of different tissues and organs, including benign and malignant tumors (See generally Oseroff, Photodynamic Therapy, Clinical Photomedicine, 387-402 (Marcel Dekker, Inc.) (1993)), early stage cancers of the lung, esophagus, stomach, cervix and cervical dysplasia, etc.. See T. J. Dougherty, Photodynamic therapy: part II, Semin. Surg. Oncol, tl, 333-334 (1995).
  • the invention discloses various phototoxic compounds and methods for utilizing such compounds, hi an embodiment, a phototoxic compound of formula (1):
  • R l5 R 2 , R 3 , P , R 5 , R , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
  • R 6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and X 1 is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid.
  • a phototoxic compound of formula (2) is a phototoxic compound of formula (2):
  • R ⁇ , R 2 , R 3 , R ⁇ , R S , R , R S and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
  • R 6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12;
  • X 2 is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid;
  • Y is one selected from the group consisting of:
  • a ls a , a 3 , a 4 , a 5 , b ls b , b 3 , and b 4 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, and hydroxyl.
  • the invention discloses a method of cleaving DNA in a sample comprising:
  • R l5 R 2 , R 3 , R t , R 5 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
  • R 6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and ⁇ is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid;
  • FIG. 1A shows structures of a peptide and a peptoid.
  • FIG. IB shows side chain residues on a peptide or peptoid.
  • FIG. 1C shows mechanism for DNA cleavage and photoxicity of a phototoxic compound.
  • FIG. 2 shows a cellular uptake of a phototoxic compound.
  • FIG 3 shows phototoxicity of phototoxic compounds.
  • FIG. 4A shows schematic synthesis of a phototoxic compound.
  • FIG. 4B shows synthesis of TQ Q and TOz derivatives.
  • FIG. 4C shows synthesis of TQ Q and TOz trimethine derivatives.
  • FIG. 5 shows structures of TO and TO-dipeptide conjugates.
  • FIG. 6 shows photocleavage of pUC18 plasmid DNA by TO-dipeptide conjugates.
  • FIG. 7 shows effect of different agents on phototocleavage of DNA by TO- K.
  • FIGS. 8A-8C show differential phototoxicity of nuclear and mitochondrial oxidants.
  • FIG. 9 A shows various TO peptidoconjugates of the invention.
  • FIG. 9B shows photocleavage of pUC18 plasmid DNA by TO peptidoconjugates analyzed by agarose gel electrophoresis.
  • FIGS. 10A-10C show confocal-microscopy images of unfixed living HeLa cells incubated with TO-Tat peptidoconjugates.
  • FIGS. 11 A-l IF show localization profiles of TO-Tat peptidoconjugates.
  • FIG. 12 shows a graph representing phototoxicity of TO-Tat peptidoconjugates.
  • the invention provides a phototoxic compound comprising a photosensitizer conjugated to a peptide or peptoid moiety.
  • the phototoxic compound mediates light-induced cleavage of DNA in a biological system.
  • the invention further relates to the use of the phototoxic compound for inducing cytotoxic activity in a cell and for photodynamic therapy.
  • the invention discloses a phototoxic compound comprising a photosensitizer described herein that is conjugated to a peptide or peptoid described herein via a covalent bond.
  • the phototoxic compound is represented by formula (1):
  • Ri., R 2 , R 3 , j, R 5 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R 6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (1 A) or
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound is represented by formula (2):
  • R 1 ⁇ R , R 3 , Ri, R 5 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R 6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, and X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound is represented by formula (2 A) or (2B):
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (3):
  • R 10 is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound is represented by formula (3 A) or (3B):
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (4):
  • R 10 is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (4A) or
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, h an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (5):
  • R ⁇ is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (5A):
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (6):
  • R ⁇ is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (6A): wherein X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the invention discloses a method of synthesizing the phototoxic compounds of the present invention, hi an embodiment, the phototoxic compounds can be formed by a process of synthesizing the photosensitizer part and the peptide or peptoid parts, followed by conjugating the photosensitizer parts to the peptide or peptoid parts.
  • the invention discloses a pharmaceutical composition that comprises a phototoxic compound described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is administered into a cell or a subject by a variety of methods known in the art.
  • the pharmaceutical composition can be administered by methods including but being not limited to, oral, topical, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the pharmaceutically composition can be used to treat a variety of diseases or abnormal conditions.
  • a light source for irradiation of the photosensitizers of the present invention depends on the type of the tissue or cell and the location of the tissue or cell in a subject.
  • the light is from ultraviolet to visible and infrared red light.
  • a phototoxic compound refers to a substance that produces a cytotoxic effect in a cell when irradiated with electromagnetic energy of an appropriate wavelength.
  • the cytotoxic effect is through cleavage of DNA in the cell.
  • a phototoxic compound is irradiated with light of an appropriate wavelength; in an embodiment, the light is ultraviolet to visible and near infrared.
  • a phototoxic compound normally comprises a photosensitizer.
  • a phototsensitizer produces singlet oxygen (an excited form of energy) upon irradiated with proper energy level and wavelength. The singlet oxygen is highly reactive and readily oxidizes a target molecule. Such a mechanism occurring in a cell, is highly cytotoxic.
  • a phototoxic compound comprises a photosensitizer conjugated to a moiety.
  • the moiety comprises a polymer.
  • the moiety comprises a peptide or peptoid that facilitates cleaving DNA in a cell.
  • a phototoxic compound that comprises a photosensitizer and a peptide or peptoid a singlet oxygen produced by the photosensitizer reacts with the functional group on the peptide or peptoid, to form an active peroxide.
  • the active peroxide reacts with DNA and cleavages DNA.
  • the functional group includes a side chain residue of an amino acid.
  • the side chain residue is from an aromatic amino acid, or a derivative thereof.
  • peptide refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. See FIG. 1 A. It also refers to either a full-length naturally-occurring amino acid sequence or a fragment thereof between about 2 and about 500 amino acids in length.
  • the peptide comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about
  • amino acids for example, ⁇ -alanine, phenyl glycine and homoarginine may be included. Commonly-encountered amino acids which are not gene- encoded may also be used in the present invention. In an embodiment, the amino acids may be either the D- or L- optical isomer.
  • a "peptoid" refers to a polyamide of between 2 and 500 units having one or more substituent on the amide nitrogen atom. See FIG. 1 A.
  • a peptoid is a synthetic analog of a peptide with the difference being that while a side-chain residue on a peptide is attached to a carbon atom ⁇ - to the carbonyl group, in a peptoid, the "side-chain residue" is attached to the amide nitrogen atom.
  • Peptoids are synthetic polymers with controlled sequences and lengths, that can be made by automated solid-phase organic synthesis to include a wide variety of side-chains having different chemical functions.
  • the peptoid comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85
  • peptoids can include a wide variety of different, non-natural side-chain residues because in peptoid synthesis the R group can be introduced as a part of an amine or by alkylation of the amine or the amide nitrogen. This is in contrast to synthetic peptides for which the incorporation of non-natural side-chain residues requires the use of non-natural ⁇ - protected amino acids.
  • Peptoids can be synthesized in a sequence- specific fashion using an automated solid-phase protocol, e.g., the sub- monomer synthetic route. See, for example, Wallace et al., Adv.
  • the term "transferring," when used in reference to a drug or agent or a phototoxic compound, means the addition of the drug or agent to an assay mixture, or to a cell in culture.
  • the term also refers to the administration of the drug or agent or phototoxic compound to an organism, preferably an animal, or a human.
  • Such administration can be, for example, by injection (in a suitable carrier, e.g., sterile saline or water) or by inhalation, or by an oral, transdermal, rectal, vaginal, or other common route of drug administration.
  • a "subject" refers to any human or non-human organism.
  • sample refers to any plant, animal or viral material containing DNA or RNA, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment.
  • the sample containing nucleic acids can be drawn from any source and can be natural or synthetic.
  • the sample containing nucleic acids may contain of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or copolymers of deoxyribonucleic acids and ribonucleic acids or combinations thereof.
  • the sample may have been subject to purification (e.g. extraction) or other treatment.
  • the term “sample” can also refer to "a biological sample.”
  • a biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • a biological sample further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.
  • the sample has been removed from an animal, but the term "biological sample” can also refer to cells or tissue analyzed in vivo, i.e., without removal from animal.
  • a biological sample will contain cells from the animal, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure the cancer-associated polynucleotide or polypeptides levels.
  • a biological sample further refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • a “therapeutic composition” refers to a composition that upon delivered into a cell or a subject, acts upon the cell or subject to correct or compensate for an underlying molecular deficit, or counteract a disease state or syndrome of the cell.
  • cytotoxic activity refers to effects or activities that facilitate reduction of host cell viability, including cell death. Such effects and activities may be associated with, for example, induction of apoptosis in the host cell, reduction of host cell protein synthesis, reduction in host cell transcription, genomic DNA fragmentation, membrane disintegration, breakdown of the nuclear lamina, change in potential of a cell and the like.
  • cleaving refers to a non-specific and specific fragmentation of a biopolymer including nucleic acids and peptides etc.
  • the invention discloses a phototoxic compound that includes a photosensitizer conjugated to a peptide or peptoid moiety.
  • the phototoxic compound cleavages DNA in a biological system upon irradiation by light.
  • the phototoxic compound is used in a photodynamic therapy for treatment of diseases.
  • a photosensitizer is a substance which upon irradiation with light, absorbed energy and induces a biological effect, for example, a cytotoxic effect.
  • Each type of photosensitizer is activated with a typical wavelength of light.
  • a photosensitizer produces singlet oxygen upon irradiation with light at the proper energy level and wavelength.
  • the photosensitizer is converted to an energized form that can react with atmospheric oxygen such that, upon decay of the photosensitizer to the unenergized/background state, singlet oxygen is produced.
  • Singlet oxygen is highly reactive and is toxic to a proximal target.
  • a photosensitizer used should have a sufficiently low toxicity to a cell to permit administration to a subject with a medically acceptable level of safety. As such, the photosensitizer should not be toxic absent irradiation with energy of appropriate wavelength.
  • a photosensitizer of the invention should be readily soluble in a variety of solvents, including those in which it is coupled to the peptide or peptoid moiety and those in which it is administered to a subject. Those of ordinary skill in the art will recognize what is desirable solubility depending on the conditions in which the photosensitizer is coupled to form a conjugate (or the conditions in which the conjugate is administered).
  • the photosensitizer and targeting moiety can be coupled in a reaction requiring solubility in DMSO, water, ethanol, or a mixture thereof (e.g., a 1:1 mixture of DMSO: H 2 O or 5% ethanol in water).
  • DMSO solubility in water
  • ethanol e.g., a 1:1 mixture of DMSO: H 2 O or 5% ethanol in water.
  • Various photosensitizers are known and can be used in the practice of this invention.
  • Photosensitizers typically have chemical structures that include multiple conjugated rings that allow for light absorption and photoactivation. They differ in properties of light absorption and fluorescence, biodistribution, temporal uptake, and mechanisms of photoactivatable cytotoxicity.
  • photosensitizers can include, but are not limited to, hematoporphyrins, such as hematoporphyrin HC1 and hematoporphyrin esters (Dobson et al., Arch. OralBiol. 37:883-887 (1992)); dihematophorphyrin ester (Wilson et al., Oral Microbiol. Immunol.
  • hematoporphyrin IX and its derivatives (Russell et al., Can. J. App. Spectros. 36:103-107, available from Porphyrin Products, Logan, Utah); 3,1-meso tetrakis (o- propionamidophenyl) porphyrin; hydroporphyrins such as chlorin, herein, and bacteriochlorin of the tetra (hydroxyphenyl) porphyrin series, and synthetic diporphyrins and dichlorins; o-substituted tetraphenyl porphyrins (picket fence porphyrins); chlorin e6 monoethylendiamine monamide (CMA; Goff et al.
  • CMA chlorin e6 monoethylendiamine monamide
  • Other potential photosensitizer can include but are not limited to, pheophorbides such as pyropheophorbide compounds, anthracenediones; anthrapyrazoles; aminoanthraquinone; phenoxazine dyes; phenothiazine derivatives; chalcogenapyrylium dyes including cationic selena- and tellura-pyrylium derivatives; verdins; purpurins including tin and zinc derivatives of octaethylpurpurin and etiopurpurin; benzonaphthoporphyrazines; cationic imminium salts; and tetracyclines.
  • pheophorbides such as pyropheophorbide compounds, anthracenediones; anthrapyrazoles; aminoanthraquinone; phenoxazine dyes; phenothiazine derivatives; chalcogenapyrylium
  • the invention discloses a phenyl-xanthenone-based photosensitizer having a structure represented by formula (A):
  • Ri, R 2 , R 3 , ⁇ , R 5 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R 6 is selected from the group consisting of hydroxyl and alkoxy, and R is a moiety conjugated to the photosensitizer.
  • R comprises a polymer.
  • R comprises a peptide or peptoid comprising at least one amino acid or amino acid side chain residue. More preferably, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the photosensitizer is 2,4,5,7-Tetraiodo-6-methoxy-9-(2,3,4,5- tetrachloro-phenyl)-xanthen-3 -one-based photosensitizer having a structure represented by
  • the photosensitizer is 2,4,5,7-Tetraiodo-6-methoxy-9-phenyl- xanthen-3-one having a structure represented by (A 2 ):
  • the photosensitizer is a benzothiazolyl-dihydroquinoline-based photosensitizer having a structure represented by formula (B):
  • R is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, and R is a moiety conjugated to the photosensitizer.
  • R comprises a polymer.
  • R comprises a peptide or peptoid comprising at least one amino acid or amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the photosensitizer is 4-(3-Methyl-benzothiazol-2-ylmethylene)- 1,4-dihydro-quinoline having a structure of (Bi):
  • the photosensitizer is 4-[3-(3-Methyl-benzothiazol-2-yl)- allylidene]-l,4-dihydro-quinoline having a structure of (B 2 ):
  • invention discloses a pyridinylidene-benzothiazole-based photosensitizer having a structure represented by formula (C):
  • R" is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, and R is a moiety conjugated to the photosensitizer.
  • R comprises a polymer.
  • R comprises a peptide or peptoid.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the photosensitizer is 3-Methyl-2-[3-(lH-pyridin-4-ylidene)- propenylj-benzothiazole having a structure of (Ci):
  • the photosensitizer of the present invention is conjugated to either a peptide or a peptoid (See FIG. 1 A) to form a phototoxic compound.
  • the peptide or peptoid is covalently conjugated to a photosensitizer via the amide group on the amino acid backbone, an embodiment, the peptide or peptoid comprises between about 1 and about 500 amino acid units in length, and can be the natural L-enantiomer, or the unnatural D-enantiomer, or a D- and L-enantiomer mixture.
  • the peptide or peptoid length is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about
  • the peptide or peptoid comprises at least one side-chain residue selected from aromatic amino acids (i.e., Phe, Tyr, Trp).
  • the side-chain residue from the aromatic amino acids is located proximate to the photosensitizer, i.e., about zero, or about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight amino acid units away from the photosensitizer on the peptide.
  • the peptide or peptoid comprises side-chain residues from Table 1.
  • At least one amino acid side-chain residue on the peptide or peptoid is substituted with a non-amino acid side-chain residue.
  • the non- amino acid side-chain residue is a aryl group.
  • aryl refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes without limitation carbocyclic aryl, aralkyl, heterocyclic aryl, biaryl groups and heterocyclic biaryl etc.
  • the non-amino acid side-chain reside is an indole- based, or a phenol-based, or an imidazole-based group (See FIG. IB.).
  • the substitution occurs at the amino acid unit that is about 0 to about 4 units away from the photosensitizer. h an embodiment, the substitution occurs at the unit most proximate (0 unit away from the photosensitizer) to the photosensitizer. hi an embodiment, further substitutions of the side-chain residue can be introduced for purposes of increased solubility, decreased aggregation and altered extent of hydrophobicity.
  • an embodiment provides a phototoxic compound comprising a photosensitizer described herein that is conjugated to a peptide or peptoid described herein via covalent bond.
  • the phototoxic compound can be represented by formula (1):
  • R ls R , R , R 4 , R 5 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R 6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (1 A) or
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (2):
  • R ls R 2 , R 3 , R ⁇ R 5 , R 7 , R 8 and R 9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R 6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, and X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (2A) or (2B):
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof
  • Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (3):
  • R 10 is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12
  • Xi is apeptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (3 A) or
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (4):
  • R 10 is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from FIG. IB.
  • the phototoxic compound can be represented by formula (4A) or
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from FIG. IB.
  • the phototoxic compound can be represented by formula (5):
  • R ⁇ is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12
  • Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phtotoxic compound can be represented by formula (5A):
  • X 1 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
  • the phototoxic compound can be represented by formula (6):
  • R ⁇ is either hydrogen or alkyl group
  • m is 1, or 2, or 3, or 4
  • n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12
  • X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue.
  • the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
  • the phototoxic compound can be represented by formula (6A): wherein X 2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, hi an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from FIG. IB.
  • the phototoxic compounds of the present invention are stable at room temperature and soluble in water and polar non-aqueous medium.
  • This water solubility indicates that the phototoxic compounds of the present are able to form a soluble formulation with a pharmaceutically acceptable carrier and functionally interact with various tissues or cells, which have aqueous environment.
  • the stability indicates that these phototoxic compounds are able to maintain their activity for a long time upon photo-induced activation, making these phototoxic compounds a very efficient agent for disease treatments.
  • the phototoxic compounds exhibit low cytotoxicity because they will not cause or contribute to an adverse biological reaction inside a tissue or cell due to their water solubility.
  • the phototoxic compounds induce cytotoxic activity in a cell upon irradiation with light of proper energy and wavelength
  • the phototoxic compound acts in the cell via a mechanism of DNA cleavage.
  • the photosensitizer on the phototoxic compound absorbs light energy to cause a promotion of the photosensitizer from its ground state to the extremely unstable excited singlet state with a half-life in range of 10 "6 to 10 "9 seconds.
  • the singlet excited photosensitizer either decays back to the ground state, resulting in the fluorescence or undergoes intersystem crossover to the longer lived (10 "3 second) tripled excited state.
  • the triplet photosensitizer reacts with ground state oxygen to produce singlet oxygen ( O 2 ) while the triplet photosensitizer turns back to its ground state.
  • the singlet oxygen then reacts with different biomolecules to cause oxidative damages.
  • the singlet oxygen reacts with a side-chain residue on the conjugated peptide or peptoid moiety to form a highly active peroxide on the side-chain residue (See Scheme 1 (FIG. IC).)
  • FIG. IC shows Scheme 1 which is a mechanism for DNA cleavage and phototoxicity of dye (S) - peptide conjugates.
  • the highly active peroxide then serves as scissors to directly cleave DNA and to prevent the DNA replication.
  • the phototoxic compounds possesses several advantages over those found in the prior art.
  • the advantages include low toxicity, high stability and targeted cleavage of DNA, etc.
  • the singlet oxygen produced by the prior art photosensitizers directly oxidizes a substrate in a cell.
  • the substrate can be different types of proteins, or lipids, or RNAs, or DNAs.
  • Goosey et al. reported that a direct reaction between a singlet oxygen and a susceptible proteins results in non-disulfide covalent cross-links in the proteins. See Goosey et al., "Cross-linking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen," Science, 208(4449): 1278-80 (June 13, 1980).
  • cytotoxic effect a cytotoxic effect on the cell.
  • necrosis refers to cell death in living tissues characterized by breakdown of cell membranes. Necrosis is always pathological. In necrosis, death of a large number of cells in one tissue area occurs as opposed to a method of selective cell death (apoptosis). Apoptosis is also known as programmed cell death. It can occur in normal tissues, for example, as a means of regulating cell numbers, and during embryological development. It is also seen in pathological processes.
  • Apoptosis is brought about by a complex system of cell signaling pathways and enzyme events. Morphologically, apoptic cells shrink and the nucleus condenses. The organelles and the nucleus break up and the cells break into fragments.
  • the light source for irradiation of the photosensitizers depends on the type of the tissue or cell and the location of the tissue or cell in a subject. In an embodiment, the light is from ultraviolet to visible and infrared red light. In an embodiment, the light is visible light. In an embodiment, laser light source can be used to provide the exact selection of wavelengths and the precise application of light.
  • the laser light sources include, but is not limited to, pulsed lasers such as the gold vapor laser and the copper vapor laser-pumped dye laser, tunable solid-state lasers such as the neodymium: YAG laser, and the semiconductor diode lasers etc.
  • the phototoxic efficiency with respect to oxidation of a target molecule by a phototoxic compound can be determined by both in vitro and in vivo assays well known in the art.
  • an in vitro assay can include measuring the ability of the phototoxic compound to "bleach" the substrate.
  • An in vivo assay with a cell can include evaluating DNA degradation in the cell or apoptosis of the cell in the presence of the irradiated and unirradiated phototoxic compound, hi an embodiment, the apoptosis can be determined by measuring survival of the cell, hi an embodiment, the apoptosis can be determined by measuring DNA synthesis.
  • the invention discloses a method of synthesizing a phototoxic compound.
  • the phototoxic compounds can be formed by a general process of synthesizing the photosensitizer part and the peptide or peptoid parts, followed by conjugating the photosensitizer parts to the peptide or peptoid parts.
  • Peptides can be synthesized by different methods well known in the art, including ribosomally-directed fermentation methods, as well as non- ribosomal strategies and chemical synthesis methods, h an embodiment, peptides containing the 20 natural amino acids and those greater than about 30 residues can be prepared via recombinant expression systems that utilize the ribosomally directed peptide synthesis machinery of a host organism, e.g., E. coli. In an embodiment, smaller peptides (less than 30 residues) and peptides which contain unnatural or non-proteinogenic amino acids or modified amino acid side chains often prepared through a more general solution-phase chemical synthesis of peptides (e.g., using N- Boc protection and the activated ester route).
  • Protocols for sequence solution-phase chemical synthesis of peptides have been described in Andersson et al., Biopolymers 55:227-250 (2000).
  • One current method used for generating peptides is solution-phase chemical synthesis, which employs a N-tert-butoxy (N-Boc) protected amino acid and a C-protected amino acid (Andersson et al., Biopolymers 55: 227-250 (2000)).
  • N-Boc N-tert-butoxy
  • An alternative solution- phase method for chemically-catalyzed peptide synthesis employs pre-activated esters as the carboxyl component for coupling (Andersson et al., Biopolymers 55: 227-250 (2000)).
  • Solid- phase peptide synthesis uses insoluble resin supports, and has simplified and accelerated peptide synthesis and facilitated purification (Merrifield, R.B., J. Am. Chem. Soc. 85: 2149-2154 (1963)). Since the growing peptide is anchored on an insoluble resin, unreacted soluble reagents can be removed by simple filtration or washing without manipulative losses.
  • solid phase peptide synthesis can be performed using automation.
  • a peptoid can be synthesized by a similar method to the synthesis of a peptide described above. For example, synthesis of a peptoid can be carried out by methods described in Murphy, J.
  • a photosensitizer-peptide conjugates i.e., phototoxic compound
  • the photosensitizer bearing an electrophilic moiety reacts with a nucleophilic group, i.e., amino terminus on a peptide or peptoid.
  • the method can use commercially available Rink amide resin on a solid support for coupling the photosensitizer to the peptide or peptoid. Yield from coupling reactions can be assessed by spectroscopy.
  • couplings can be performed using 4 equivalents of Fmoc protected amino acid, 4 equivalents of HBTU and 8 equivalents of Hunig's base in DMF for 3 hours.
  • deprotection of the Fmoc group can be achieved using 20% piperidine in DMF for 30 minutes (to minimize diketopiperazine formation, dipeptides were deprotected using 50% piperidine in DMF for 5 min).
  • the dye moiety is attached to a resin-bound peptide as described below.
  • the dye-peptide conjugates are simultaneously deprotected and cleaved from the resin with a 95:5 TFA: ⁇ PS solution.
  • a pharmaceutical composition that comprises a phototoxic compound described herein and a pharmaceutically acceptable carrier.
  • solid or liquid pharmaceutically acceptable carriers can be employed.
  • Solid carriers include but are not limited to, starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include but are not limited to, syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • a solution such as an ampoule
  • aqueous or nonaqueous liquid suspension aqueous or nonaqueous liquid suspension.
  • the pharmaceutical composition can be used in its solid form or dissolved in an appropriate solvent for addition to the carrier (solid or liquefied) or dissolved in an appropriate solvent. Mixtures should be in appropriate solvents for dissolving both medicament and carrier, and at the desired degree of medicament purity.
  • the phototoxic compound and the carrier upon hydration, at the appropriate pH for the pharmaceutical composition, form a complex which facilitates delivery of the phototoxic compound to a target.
  • other additives and pharmaceutical excipients can also be added, during or after formulation, to improve the ease of formulation, formulation stability, speed of reconstitution, delivery of the formulation. These include, but are not limited to, penetration enhancers, targeting aids, anti-oxidants, preservatives, buffers, stabilizers, solid support materials.
  • the composition may include osmoregulators if required, such as but not limited to, physiologically buffered saline (PBS), carbohydrate solution such as lactose, trehalose, higher polysaccharides, or other mjectable material.
  • PBS physiologically buffered saline
  • carbohydrate solution such as lactose, trehalose, higher polysaccharides, or other mjectable material.
  • stabilizers A wide variety of excipients and stabilizers are known in the art and their use will depend on formulation type and application requirements.
  • the function of stabilizers is to provide increased storage stability in cases where the phototoxic compound or carrier is labile to heat, cold, light or oxidants or other physical or chemical agents.
  • Other purposes for stabilizers can be for maintaining phototoxic compound and/or carrier in a form appropriate for transport to and uptake at the target site.
  • the excipients or stabilizers can be added either prior to deposition step or after the hydration step.
  • the pharmaceutical composition can be formulated into dosage forms such as capsules, impregnated wafers, ointments, lotions, inhalers, nebulizers, tablets, or mjectable preparations.
  • the pharmaceutically composition can be used to treat a variety of diseases or abnormal conditions.
  • diseases include but are not limited to malignancies and inflammatory diseases, such as multiple types of cancer or tumor at different tissues and organs, demiatological diseases, atherosclerosis, infectious diseases, theumatoid arthritis, age-related macular degeneration, restenosis, ALDS, hematological diseases, etc.
  • the pharmaceutical composition can be administered by a variety of methods known in the art.
  • the route and/or mode of administration will vary depending on the conditions of target organisms and the desired results.
  • the pharmaceutical composition can be administered by methods including but being not limited to, oral, topical, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • Effective amounts or doses of the composition for treating a disease or condition can be determined using recognized in vitro systems or in vivo animal models for the particular disease or condition.
  • One of the factors that determine the dosage is the irradiation time. If it is desired to irradiate only for short time, the concentration of the composition can be increased.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dosage can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • compositions can be tested for inhibition of tumor cell growth in cell culture using standard assays with any of a multitude of tumor cell lines of human or nonhuman animal origin.
  • Many of these approaches, including animal models, are described in detail in Geran, R. I. et al., "Protocols for Screening Chemical Agents and Natural Products against Animal Tumors and Other Biological Systems (Third Edition),” Cane. Chemother. Reports, Part 3, 3:1-112.
  • a typical single dosage for administration to cells comprises between about 1 ng and about 500 mg of the active phototoxic compound per 1 x 10 5 cells, preferably between about 100 ng and about 100 mg of the active phototoxic compound per 1 x 10 5 cells.
  • the dosage for administration to a subject comprises between about 1 ng and about 10 g of the active phototoxic compound per kilogram body weight, preferably between about 0.1 mg and about 100 mg per kilogram body weight.
  • Methods for irradiation include, but are not limited to, the administration of laser, nonlaser, or broad band light. Irradiation can be produced by extracorporeal or intraarticular generation of light of the appropriate wavelength, hi an embodiment, light used in the invention can be administered using any device capable of delivering the requisite power of light including, but not limited to, fiber optic instruments, arthroscopic instruments, or instruments that provide transillumination. Delivery of the light to a recessed, or otherwise inaccessible physiological location can be facilitated by flexible fiber optics (implicit in this statement is the idea that one can irradiate either a broad field, such as the lung or a lobe of the lung, or a nan-ow field where bacterial cells may have localized). Examples
  • the synthesis of formula (1) was performed by the following method.
  • the rose bengal moiety (RB) was attached by treating a resin-bound peptide (1.0 equiv.) with rose bengal 5-carboxy pentyl ester (3.0 equiv.), HBTU (3.0 equiv.), HOBt (3.0 equiv.) and DIPEA (6.0 equiv.) in DMF for about 12 hours.
  • the synthesis of formula (2) was performed by the following method.
  • the erythrosin moiety (ER) was attached via an aminohexanoic acid spacer by treating a resin-bound peptide (1.0 equiv.) with erythrosin B isothiocyanate isomer II (1.0 equiv) and DIPEA (5 equiv.) in DMF for about 12 hours.
  • One derivative featured a carboxylate-terminated tether attached to the quinoline nitrogen (TO Q , 3), and the other featured the same linker attached to the benzothiazole nitrogen (TO z , 6).
  • Derivatives of quinoline and benzothiazole were modified with 11 -bromoundecanoic acid (selected to minimize cyclization reactions that occurred with shorter linkers for TOz) and subsequently coupled with the appropriate heterocyclic quaternary salt to yield the cyanine dye containing a carboxylate functionality.
  • Derivative 9 was synthesized by alkylation of one equivalent of 2-methylbenzothiazole with three equivalents of iodomethane in refluxing dioxane and subsequently reacted with conjugated NN'-diphenylformamidine in refluxing acetic anhydride to give acetanilide derivative 9 which was coupled to either derivative 7 or 8 to yield the trimethine cyanine dye containing a carboxylate functionality.
  • the thiazole orange moiety (TO) was attached by treating a resin- bound peptide (1.0 equiv.) with thiazole orange derivative (4.0 equiv.), HBTU (4.0 equiv.), and DIPEA (8.0 equiv.) in DMF for about 3 hours.
  • Peptoids were synthesized according to the method of Zuckerman. 4"6
  • the Fmoc-Rink amide resin (1.0 equiv.) was treated with 20% piperidine in DMF for about 30 minutes.
  • the free resin-bound amine was then treated with a solution of bromoacetic acid (10 equiv.) and diisopropylcarbodiimide (10 equiv.) in DMF for about 30 minutes. This procedure was repeated.
  • the resin was then treated with a solution of primary amine (40 equiv.) in DMF for about 12 hours. These two steps were repeated until an oligomer of desired length was obtained.
  • the resin was then treated with Fmoc-protected amino acid (4.0 equiv.), HBTU (4.0 equiv.), and DIPEA (8.0 equiv.) in DMF for about 3 hours.
  • the dye moiety was attached to the resin as described earlier.
  • the dye-peptoid conjugates were simultaneously deprotected and cleaved from the resin with a 95:5 TFA : TIPS solution.
  • the solution was concentrated in vacuo and purified via RP-HPLC (H 2 O/CH 3 C ⁇ in 0.1% TFA).
  • the products were isolated by lyophilization and characterized by MALDI-TOF mass spectrometry.
  • the purity of the peptides was > 95% as determined by analytical RP-HPLC (H 2 O/CH 3 CN in 0.1% TFA).
  • Example 4 DNA-binding characterization of Thiazole Orange (TOVPeptide Conjugates
  • Solvents were purchased from Fisher and reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Acros Organics (Morris Plains, NJ). Amino acids were purchased from Advanced ChemTech (Louisville, KY). Calf thymus DNA (CT DNA) was purchased from Sigma (St. Louis, MO). All solvents and reagents were used without further purification. HPLC grade acetonitrile and Millipore water were used for HPLC analysis. The buffer used in all experiments was 50 mM sodium phosphate, 10 mM sodium chloride (pH 7). Reversed-phase HPLC was performed using a HP 1100 system with a Varian 250 x
  • UV analysis was performed on a Hewlett Packard 8452A Diode Array Spectrophotometer. Steady state fluorescence measurements were performed on a Jobin Yvon Horiba Fluorolog®-3. For all steady state measurements the 490- 650 run. Dissociation constants were measured using a Perkin Elmer Wallac Victor Fluorescence reader fitted with a 450-490 nm excitation filter and a 515 nm long pass emission filter. Hypochromicity measurements
  • TO-peptide conjugates were synthesized on solid support using commercially available Wang-Fmoc-Lys(Boc) (0.7 mmol/g, Advanced ChemTech). Couplings were performed using 4 equivalents of Fmoc protected amino acid, 4 equivalents of HBTU and 8 equivalents of diisopropylethylamine in DMF for about 3 hours. Deprotection of the Fmoc group was achieved using 20% piperidine in DMF for about 30 minutes (after coupling of the first amino acid to the resin, Fmoc deprotection was achieved using 50% piperidine in DMF for about 5 minutes in order to minimize diketopiperazine formation).
  • TO-peptide conjugates were simultaneously deprotected and cleaved from the resin with a solution of 9:1:1:1 trifluoracetic acid: triethylsilane: triisopropylethylamine: H 2 O for about 30 minutes.
  • the solution was concentrated under reduced pressure in the presence of toluene in order to remove any residual TFA.
  • TO-peptide conjugates were dissolved in a minimal amount of methanol, which was then removed under vacuum to yield a red solid.
  • Solvents were purchased from Fisher and reagents were purchased from Aldrich Chemical Co. or Acros Organics. Amino acids were purchased from Advanced ChemTech. Both solvents and reagents were used without further purification.
  • Reverse phase HPLC was performed on a Varian 250 x 4.6 mm stainless steel column packed with Microsorb-MV 300 C18 (5 mm). A flow rate of about 1.0 mL/min. was used with an aqueous solution buffered with 50 mM ammonium acetate and a linear gradient from 20 to 100% acetonitrile over 80 min. ⁇ H NMR spectra were recorded on a Varian 400 MHz spectrometer.
  • FIG. 7A percent cleaved values represent averages of 5-10 replicates, and a correction factor of 1.22 was used to adjust for the decreased stainability of supercoiled DNA.
  • the data shown in FIG. 7A was obtained with 10 mM NaNs and 200 mM trolox. These scavenger concentrations produced minimal fluorescence quenching for DNA-bound TO-WK.
  • FIG. 7A shows the effect of different agents on photocleavage of DNA by TO-WK; 5 minute irradiations were performed as described to quantitate changes in efficiency relative to TO-WK in buffered H 2 O under ambient conditions.
  • FIG. 7B shows time dependence of Trp- based peroxide formation upon irradiation of TO-WK in 70% glycerol-30% X 2 O measured using a modified FOX assay.
  • the procedure for the modified FOX assay was based on that developed by Gebicki and co workers and described in ref. 15. 50 ⁇ M TO or TO-WK was added to a solution of 70% glycerol and 30% H2O or D2O in the dark. Samples were irradiated for 0, 15, 35, 45, or 60 minutes with a 365 nm transilluminating lamp. After irradiation, one volume of glacial acetic acid was added, followed by 700 ⁇ M xylenol orange and 280 ⁇ M ammonium iron(lT) sulfate hexahydrate. 50 ⁇ M H2O2 was added to a solution of 70% glycerol to provide a standard.
  • FIG. 5 illustrates the series of compounds synthesized and tested for DNA photocleavage activity.
  • the conjugates feature thiazole orange (TO), a fluorescent DNA intercalator, ⁇ conjugated to synthetic dipeptides through a linker attached to the quinoline nitrogen of the heterocycle.
  • TO thiazole orange
  • TO-GK glycine
  • TO-YK tyrosine
  • TO-WK tryptophan
  • FIG. 6 A shows photocleavage of pUC18 plasmid DNA by TO dipeptide derivatives analyzed by agarose gel electrophoresis. Solutions contained 20 ⁇ M TO or TO-peptide conjugate, 75 ⁇ M (bp) pUC18, and 25 mM sodium phosphate (pH 7).
  • FIG. 6B shows time dependence of photocleavage activity for TO, TO-dipeptides and a WK dipeptide. Irradiation of DNA in the presence of a high concentration of a WK dipeptide or TO did not result in strand scission (FIG. 6B). These experiments indicated that the reaction observed required both TO and W.
  • a TO-YK conjugate also produced DNA cleavage upon photoexcitation, although with lower efficiency than TO-WK (FIG. 6B).
  • the observation of activity for TO-WK and TO-YK that was significantly higher than for TO-GK indicates that the aromatic amino acids form reactive species in the presence of the TO excited state that are not accessible with the aliphatic amino acid.
  • a subset of naturally-occurring amino acids is known to react with singlet oxygen to form peroxides.
  • Tip and Tyr efficiently react with 1 O 2 , with quenching rate constants (k iot ) of 3.2 x 10 7 and 0.5 x 10 7 M ⁇ s -1 , respectively.
  • Gly exhibits very low reactivity upon exposure to ⁇ 2, with k to t ⁇ 0.1 x 10 7 M ⁇ s -1 .
  • the trend in the rate constants coincides with the DNA cleavage activity of the TO-peptide conjugates.
  • trolox a peroxyl-radical scavenger
  • Figure 7A To test directly whether peroxides were formed upon irradiation of the TO-peptide conjugates, a modified FOX assay was employed.— This analysis showed significant levels of peroxide formation upon irradiation of TO-WK that increased when D 2 O was introduced into the samples. These results are consistent with the production of amino-acid based peroxides formed by a reaction with O 2 generated by photoexcited TO.
  • a previous report of DNA cleavage by thermally- generated peroxides provides a precedent for strand scission by this class of chemical species. 1 ⁇
  • the DNA-binding peptide-intercalator conjugates described here exhibit DNA cleavage activity that appears to result from the reaction of ! O with amino acids. Damage to protein side chains is proposed to be a potential source of the deleterious effects of O 2 — The discovery of a model system that permits the photogeneration of ! O in proximity to reactive residues will facilitate studies of this damage pathway. Moreover, TO-peptide conjugates will provide useful tools for analysis of the chemical reactions of amino acid- based peroxides with DNA.
  • the K ⁇ values corresponding to TO, TO-GK, TO-WK, and TO-YK bound to calf thymus (CT) DNA were 1.8 ⁇ 0.1, 2.1 ⁇ 0.1, 2.4 ⁇ 0.3, and 2.9 ⁇ 0.2 ⁇ M, respectively.
  • the quantum yields of TO, TO-GK, TO- WK, and TO-YK bound to CT DNA were 0.11,4 0.20 ⁇ 0.02, 0.16 ⁇ 0.02, 0.22 ⁇ 0.01. See ESIf for procedures and conditions used to obtain these values.
  • HeLa cells were incubated with 10 ⁇ M TO-peptide conjugates (phototoxic compounds) as shown in FIG. 3.
  • the HeLa cells together with the phototoxic compounds were irradiated with visible light for about 10 minutes and incubated for about 24 hours before counting the cells for apoptosis or survival rate.
  • Fluorescence imaging of HeLa cells incubated with TO-peptide conjugates demonstrated cellular uptake of the phototoxic compound (See FIG. 2).
  • FIG. 2 shows fluorescence imaging of HeLa cells incubated with TO-tat conjugate demonstrating cellular uptake.
  • the compounds such as TO-WK and TO-WRKKRRQRRR formed peroxides (See FIG. 7B) and exhibited significant and selective phototoxicity.
  • Oxidative stress resulting from the intracellular release of chemical oxidants or free radicals is known to exert deleterious effects on biological function.
  • exposure to light or sensitizers can produce singlet oxygen ( ! O 2 ), a highly reactive, mutagenic, and genotoxic species that induces oxidative stress.
  • An explanation for such DNA-cleavage activity is an identified subset of protein residues (including tryptophan, cysteine, histidine, and tyrosine) which react with 2 O 2 to form peroxides.
  • Peroxyl radicals are reasonable candidates as the active DNA-cleaving species, as precedent exists for strand scission through hydrogen abstraction from the DNA backbone by thermally-generated peroxides. Indeed, studies of TO peptidoconjugates displaying strand- scission activity revealed that amino acid based peroxides are the active species that induce DNA cleavage.
  • TO peptidoconjugates that access human cells and exhibit amino-acid-dependent phototoxicity.
  • the TO conjugates feature a portion (residues 49-57) of the HIV-1 transactivator of transcription (Tat) peptide sequence, which was previously used by other research groups to deliver appended cargoes into cells, investigated herein is whether cell-permeable TO peptidoconjugates are toxic to human cells upon photoexcitation, and to determine if toxicity can be triggered by the presence of specific amino acids.
  • a TO-Tat peptidoconjugate containing tryptophan cleaves DNA in vitro and exhibits appreciable phototoxicity, wherea a glycine-containing analogue neither elicits significant DNA cleavage, nor causes cell death.
  • a glycine-containing analogue neither elicits significant DNA cleavage, nor causes cell death.
  • FIG. 9A and FIG. 9B show various compounds utilized in the present experiment.
  • FIG. 9A shows structures of TO peptidoconjugates: compound la shows TO-W-Tat; compound 2a shows TO-GK; and compound 2b shows TO-WK.
  • TO-D-peptide conjugates la-2b were prepared on Rink amide solid support, and the N-terminus was capped with a TO derivative by using standard solid phase Fmoc chemistry. Subsequent cleavage from the resin and purification by HPLC afforded TO peptidoconjugates la-2b.
  • the non-natural D-peptide structure was used to impart resistance to proteolytic degradation.
  • the two versions of the Tat peptide, la and lb were prepared with glycine or tryptophan residues, respectively, positioned proximal to the dye to evaluate is selective DNA cleavage and phototoxicity could be observed.
  • the TO- dipeptide conjugates 2a and 2b are analogues to L-amino acid containing conjugates and are used here as positive controls for DNA cleavage.
  • the photocleavage properties of the TO-Tat peptidoconjugates were investigated with a plasmid nicking assay. Upon irradiation with visible light, the tryptophan-containing conjugate lb caused high levels of strand scission as shown in FIG. 9B. The observation that lb yields greater DNA cleavage (induced by the tryptophan-based peroxide formed upon the production of ! O2) than la (which elicits only low-level cleavage from direct 1 O 2 - ⁇ romoted damage) indicates that these TO conjugates exhibit photoreactivity analogous to that of their L-amino acid counterparts.
  • FIG. 10A shows transmission image of cells incubated with compound lb (shown in FIG. 9A);
  • FIG. 10B shows red-fluorescence image of the same cells that illustrates both cytoplasmic and nuclear uptake of lb; and
  • FIG. 10A shows transmission image of cells incubated with compound lb (shown in FIG. 9A);
  • FIG. 10B shows red-fluorescence image of the same cells that illustrates both cytoplasmic and nuclear uptake of lb;
  • IOC shows peptidoconjugate la shows an identical internalization pattern to that of lb. Less than five percent of the cells were stained when treated with propidium iodide, a dye specific for dead cells, thus reflecting that the conditions used to evaluate uptake patterns of the peptidoconjugates did not induce cell death.
  • FIG. 11A-1 IF show localization profiles of TO-Tat peptidoconjugates.
  • FIG. llA and FIG. 1 ID show red-fluorescence image of cell stained with la.
  • FIG. 11B shows visualization of mitotracker deep red-633 staining of the mitochondria.
  • FIG. 11C shows merged image of red- and blue-fluorescence images illustrating colocahzation of la with mitotracker deep red-633.
  • FIG. 1 IE shows green-fluorescence image illustrating nucleolar staining by SYTO-85.
  • FIG. 1 IF shows merged image showing that la colocalizes with SYTO-85 in the nucleoli of these cells.
  • the cells were incubated with fresh media for 24h before viability was analyzed to allow the effects of the compounds to be assessed. Cell death was not observed if cells containing either conjugate were kept in the dark. Interestingly, the Tat peptide was observed to effectively abolish the dark toxicity of the parent TO compound, as unmodified TO causes quantitative cell death even in the absence of light.
  • HBTU equiv, Advanced ChemTech, HBTU ⁇ O- enzotriazol-l-y -N
  • the dye-peptide conjugates were detached from the resin by slurrying in TFA/CH 2 CL 2 (10% v/v) and transferred to a glass funnel with a fine sinter. The solvent was allowed to drip slowly through the resin bed and washed with TFA/CH 2 C1 2 (5% v/v) and concentrated in vacuo. Deprotection was carried out by stirring the residue in
  • HeLa 229 cells were cultured as subconfluent monolayers on cell culture plates (25 or 75 cm 2 ) with vent caps (Corning) in 1 X minimum essential ⁇ medium (Gibco) supplemented with fetal bovine serum (10% v/v, ATCC) in a humidified incubator at 37°C containing CO 2 (5%).
  • Confocal microscopy HeLa cells that had been grown to subconfluence were dissociated from the surface with a solution of ethylenediaminetetraacetic acid (EDTA, 0.53mM)/trypsin (0.05%)(2mL, Cellgro) for 15 minutes at 37°C.
  • EDTA ethylenediaminetetraacetic acid
  • FIG. 8A-8C shows differential phototoxicity of nuclear and mitochondrial oxidants.
  • FIG. 8 A shows a phototoxic compound is specific for a nucleus of a cell.
  • FIG. 8A shows the localization of the nucleus specific peptidoconjugate.
  • the nucleus specific peptidoconjugate is TO-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.
  • FIG. 8B shows a phototoxic compound is specific for a mitochondria of a cell.
  • FIG. 8B shows the localization of the mitochondria specific peptidoconjugate.
  • the mitochondria specific peptidoconjugate is TO-Phe-d-Arg-Phe-Lys.
  • FIG. 8C shows a % survival of treating cells in a mitochondria. As shown, the mitochondria specific peptidoconjugate displays a greater ability to treat mitochondria of a cell. As such, the disclosed compounds allow for localized treatment of various conditions.

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Abstract

The invention discloses various phototoxic compounds which include a photosensitizer conjugated to a peptide or peptoid. Additionally, the invention discloses a method of making and using the various phototoxic compound. Furthermore, the invention discloses using such phototoxic compound for DNA cleavage and for causing cytotoxic effect in a biological system. Additionally, the present invention features using the phototoxic compound for treating diseases in a subject.

Description

PHOTOTOXIC COMPOUNDS
FIELD
The invention relates to a phototoxic compound that includes a photosensitizer conjugated to a peptide or peptoid moiety. The phototoxic compound mediates light-induced cleavage of DNA in a biological system. The invention further relates to the use of the phototoxic compound for inducing cytotoxic activity in a cell and for photodynamic therapy.
BACKGROUND
Photodynamic therapy, also referred to as photosensitization-based therapy (PDT), photoradiation therapy, phototherapy or photochemotherapy, is a medical treatment that employs a combination of light and a photosensitizing agent to generate a cytotoxic effect of cancerous or other unwanted tissues or organisms (See Dougherty, T. J., Photodynamic Therapy — New Approaches, Semin. Surg. Oncol. 5(1): 6-16 (1989); Liberman J. Light, Medicine of the Future, Santa Fe: Bear & Co. (1991); Hopper, C, Photodynamic Therapy: A Clinical Reality in the Treatment of Cancer, Lancet Oncol. 1: 212- 219 (2000)). The widely accepted PDT mechanism is that upon irradiation, the photosensitizer successively generates active oxygen species of high reactivity that interact with target molecules. Photodynamic therapy (PDT) consists of introducing a photoactive drug into a body and subsequent illumination of cells by visible or near infrared light. In the presence of oxygen, illumination activates the drug and in turn produces reactive oxygen species which are extremely destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids, leading to cell destruction.
A large number of photosensitizing compounds have been developed for photodynamic therapy during last ten years. For example, porphyrins and their derivatives absorb light strongly in the 690-880 nm region, and have been suggested for use as photosensitizers in photodynamic therapy. See U.S. Pat. Nos. 5,268,371 and 5,272,142, European Patent Nos. 213272 and 584552. See also Jori et al, "Controlled targeting of different subcellular sites by porphyrins in tumor-bearing mice", Br J Cancer 53:615-621 (1986). Photodynamic therapy possesses high effectiveness and safety compared with the conventional chemotherapy because of its relative selectivity in most sites, its compatibility with other treatment, its repeatability, its ease of delivery etc..
Photodynamic therapy has been effective in treating multiple types of cancer, including cancers of different tissues and organs, including benign and malignant tumors (See generally Oseroff, Photodynamic Therapy, Clinical Photomedicine, 387-402 (Marcel Dekker, Inc.) (1993)), early stage cancers of the lung, esophagus, stomach, cervix and cervical dysplasia, etc.. See T. J. Dougherty, Photodynamic therapy: part II, Semin. Surg. Oncol, tl, 333-334 (1995). Moreover, numerous investigations demonstrate possible practical usefulness of photodynamic therapy in diverse disease conditions including demiatological diseases, atherosclerosis, infectious diseases, rheumatoid arthritis, age-related macular degeneration, restenosis, AIDS, hematological diseases, etc.
SUMMARY
The invention discloses various phototoxic compounds and methods for utilizing such compounds, hi an embodiment, a phototoxic compound of formula (1):
Figure imgf000003_0001
is disclosed, wherein
Rl5 R2, R3, P , R5, R , R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
R6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and X1 is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid.
In an embodiment, a phototoxic compound of formula (2):
Figure imgf000004_0001
is disclosed, wherein:
R\, R2, R3, RΛ, RS, R , RS and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
R6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12;
X2 is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid; and
Y is one selected from the group consisting of:
Figure imgf000004_0002
wherein als a , a3, a4, a5, bls b , b3, and b4 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, and hydroxyl. In an embodiment, the invention discloses a method of cleaving DNA in a sample comprising:
contacting a phototoxic compound of the formula:
Figure imgf000005_0001
wherein :
Rl5 R2, R3, Rt, R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
R6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and \ is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid;
with the sample; and
irradiating the sample and the phototoxic compound with visible light to activate the phototoxic compound.
Various other phototoxic compounds and methods are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A shows structures of a peptide and a peptoid.
FIG. IB shows side chain residues on a peptide or peptoid.
FIG. 1C shows mechanism for DNA cleavage and photoxicity of a phototoxic compound.
FIG. 2 shows a cellular uptake of a phototoxic compound.
FIG 3 shows phototoxicity of phototoxic compounds.
FIG. 4A shows schematic synthesis of a phototoxic compound.
FIG. 4B shows synthesis of TQQ and TOz derivatives. FIG. 4C shows synthesis of TQQ and TOz trimethine derivatives.
FIG. 5 shows structures of TO and TO-dipeptide conjugates.
FIG. 6 shows photocleavage of pUC18 plasmid DNA by TO-dipeptide conjugates.
FIG. 7 shows effect of different agents on phototocleavage of DNA by TO- K.
FIGS. 8A-8C show differential phototoxicity of nuclear and mitochondrial oxidants. FIG. 9 A shows various TO peptidoconjugates of the invention.
FIG. 9B shows photocleavage of pUC18 plasmid DNA by TO peptidoconjugates analyzed by agarose gel electrophoresis.
FIGS. 10A-10C show confocal-microscopy images of unfixed living HeLa cells incubated with TO-Tat peptidoconjugates. FIGS. 11 A-l IF show localization profiles of TO-Tat peptidoconjugates.
FIG. 12 shows a graph representing phototoxicity of TO-Tat peptidoconjugates. DETAILED DESCRIPTION
The invention provides a phototoxic compound comprising a photosensitizer conjugated to a peptide or peptoid moiety. The phototoxic compound mediates light-induced cleavage of DNA in a biological system. The invention further relates to the use of the phototoxic compound for inducing cytotoxic activity in a cell and for photodynamic therapy.
In an embodiment, the invention discloses a phototoxic compound comprising a photosensitizer described herein that is conjugated to a peptide or peptoid described herein via a covalent bond.
In an embodiment, the phototoxic compound is represented by formula (1):
Figure imgf000007_0001
wherein Ri., R2, R3, j, R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. Preferably, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (1 A) or
(IB):
Figure imgf000008_0001
wherein Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound is represented by formula (2):
Figure imgf000008_0002
wherein R1} R , R3, Ri, R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, and X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB. In an embodiment, the phototoxic compound is represented by formula (2 A) or (2B):
Figure imgf000009_0001
wherein X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
In an embodiment, the phototoxic compound can be represented by formula (3):
Figure imgf000009_0002
wherein R10 is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12, and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof. In an embodiment, the phototoxic compound is represented by formula (3 A) or (3B):
Figure imgf000010_0001
wherein Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, h an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (4):
Figure imgf000010_0002
wherein R10 is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, and X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB. In an embodiment, the phototoxic compound can be represented by formula (4A) or
(4B):
Figure imgf000011_0001
or
Figure imgf000011_0002
wherein X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, h an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
In an embodiment, the phototoxic compound can be represented by formula (5):
Figure imgf000011_0003
wherein Rπ is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12, and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (5A):
Figure imgf000012_0001
wherein Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (6):
Figure imgf000012_0002
wherein Rπ is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12, and X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
In an embodiment, the phototoxic compound can be represented by formula (6A):
Figure imgf000013_0001
wherein X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
In an embodiment, the invention discloses a method of synthesizing the phototoxic compounds of the present invention, hi an embodiment, the phototoxic compounds can be formed by a process of synthesizing the photosensitizer part and the peptide or peptoid parts, followed by conjugating the photosensitizer parts to the peptide or peptoid parts. In an embodiment, the invention discloses a pharmaceutical composition that comprises a phototoxic compound described herein and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition is administered into a cell or a subject by a variety of methods known in the art. For example, the pharmaceutical composition can be administered by methods including but being not limited to, oral, topical, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
The pharmaceutically composition can be used to treat a variety of diseases or abnormal conditions. In an embodiment, a light source for irradiation of the photosensitizers of the present invention depends on the type of the tissue or cell and the location of the tissue or cell in a subject. In an embodiment, the light is from ultraviolet to visible and infrared red light.
Definitions:
As used herein, a "phototoxic compound" refers to a substance that produces a cytotoxic effect in a cell when irradiated with electromagnetic energy of an appropriate wavelength. In an embodiment, the cytotoxic effect is through cleavage of DNA in the cell. Typically, a phototoxic compound is irradiated with light of an appropriate wavelength; in an embodiment, the light is ultraviolet to visible and near infrared. A phototoxic compound normally comprises a photosensitizer. A phototsensitizer produces singlet oxygen (an excited form of energy) upon irradiated with proper energy level and wavelength. The singlet oxygen is highly reactive and readily oxidizes a target molecule. Such a mechanism occurring in a cell, is highly cytotoxic. In an embodiment, a phototoxic compound comprises a photosensitizer conjugated to a moiety. In an embodiment, the moiety comprises a polymer. In an embodiment, the moiety comprises a peptide or peptoid that facilitates cleaving DNA in a cell. In a phototoxic compound that comprises a photosensitizer and a peptide or peptoid, a singlet oxygen produced by the photosensitizer reacts with the functional group on the peptide or peptoid, to form an active peroxide. In an embodiment, the active peroxide reacts with DNA and cleavages DNA. h an embodiment, the functional group includes a side chain residue of an amino acid. In an embodiment, the side chain residue is from an aromatic amino acid, or a derivative thereof.
As used herein, a "peptide" refers to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds. See FIG. 1 A. It also refers to either a full-length naturally-occurring amino acid sequence or a fragment thereof between about 2 and about 500 amino acids in length. In an embodiment, the peptide comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500 amino acids. Additionally, unnatural amino acids, for example, β-alanine, phenyl glycine and homoarginine may be included. Commonly-encountered amino acids which are not gene- encoded may also be used in the present invention. In an embodiment, the amino acids may be either the D- or L- optical isomer. As used herein, a "peptoid" refers to a polyamide of between 2 and 500 units having one or more substituent on the amide nitrogen atom. See FIG. 1 A. A peptoid is a synthetic analog of a peptide with the difference being that while a side-chain residue on a peptide is attached to a carbon atom α- to the carbonyl group, in a peptoid, the "side-chain residue" is attached to the amide nitrogen atom. Peptoids are synthetic polymers with controlled sequences and lengths, that can be made by automated solid-phase organic synthesis to include a wide variety of side-chains having different chemical functions. In an embodiment, the peptoid comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500 "side-chain residues." Peptoids have a number of notable structural features in comparison to peptides. For example, whereas the side-chain ("R") groups on biosynthetically produced peptides must be chosen from among the 20 naturally-occurring amino acids, peptoids can include a wide variety of different, non-natural side-chain residues because in peptoid synthesis the R group can be introduced as a part of an amine or by alkylation of the amine or the amide nitrogen. This is in contrast to synthetic peptides for which the incorporation of non-natural side-chain residues requires the use of non-natural α- protected amino acids. Peptoids can be synthesized in a sequence- specific fashion using an automated solid-phase protocol, e.g., the sub- monomer synthetic route. See, for example, Wallace et al., Adv. Amino Acid Mimetics Peptidomimetics, 1999, 2, 1-51 and references cited therein, all of which are incorporated herein in their entirety by this reference. Generally, when attached to a binding polymer, longer peptoids provide a higher ratio of charge/translational frictional drag (i.e., α value), than shorter peptoids. The synthesis of long peptoids can be achieved using the sub-monomer protocol.
As used herein, the term "transferring," when used in reference to a drug or agent or a phototoxic compound, means the addition of the drug or agent to an assay mixture, or to a cell in culture. The term also refers to the administration of the drug or agent or phototoxic compound to an organism, preferably an animal, or a human. Such administration can be, for example, by injection (in a suitable carrier, e.g., sterile saline or water) or by inhalation, or by an oral, transdermal, rectal, vaginal, or other common route of drug administration.
As used herein, a "subject" refers to any human or non-human organism.
As used herein, the term "sample" as used in its broadest sense, refers to any plant, animal or viral material containing DNA or RNA, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. The sample containing nucleic acids can be drawn from any source and can be natural or synthetic. The sample containing nucleic acids may contain of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or copolymers of deoxyribonucleic acids and ribonucleic acids or combinations thereof. Alternatively, the sample may have been subject to purification (e.g. extraction) or other treatment. The term "sample" can also refer to "a biological sample."
As used herein, the term "a biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "A biological sample" further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. In an embodiment, the sample has been removed from an animal, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e., without removal from animal. In an embodiment, a "biological sample" will contain cells from the animal, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to measure the cancer-associated polynucleotide or polypeptides levels. "A biological sample" further refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
As used herein, a "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
As used herein, a "therapeutic composition" refers to a composition that upon delivered into a cell or a subject, acts upon the cell or subject to correct or compensate for an underlying molecular deficit, or counteract a disease state or syndrome of the cell. As used herein, "cytotoxic activity" refers to effects or activities that facilitate reduction of host cell viability, including cell death. Such effects and activities may be associated with, for example, induction of apoptosis in the host cell, reduction of host cell protein synthesis, reduction in host cell transcription, genomic DNA fragmentation, membrane disintegration, breakdown of the nuclear lamina, change in potential of a cell and the like.
As used herein, "cleaving" refers to a non-specific and specific fragmentation of a biopolymer including nucleic acids and peptides etc.
As used herein, the singular forms "a," "an," and "the" used in the specification and claims include both singular and plural referents unless the content clearly dictates otherwise.
I. Phototoxic Compound and Phototoxic Activity
The invention discloses a phototoxic compound that includes a photosensitizer conjugated to a peptide or peptoid moiety. The phototoxic compound cleavages DNA in a biological system upon irradiation by light. In an embodiment, the phototoxic compound is used in a photodynamic therapy for treatment of diseases. Photosensitizer:
A photosensitizer is a substance which upon irradiation with light, absorbed energy and induces a biological effect, for example, a cytotoxic effect. Each type of photosensitizer is activated with a typical wavelength of light. A photosensitizer produces singlet oxygen upon irradiation with light at the proper energy level and wavelength. The photosensitizer is converted to an energized form that can react with atmospheric oxygen such that, upon decay of the photosensitizer to the unenergized/background state, singlet oxygen is produced. Singlet oxygen is highly reactive and is toxic to a proximal target. In an embodiment, a photosensitizer used should have a sufficiently low toxicity to a cell to permit administration to a subject with a medically acceptable level of safety. As such, the photosensitizer should not be toxic absent irradiation with energy of appropriate wavelength. In addition, a photosensitizer of the invention should be readily soluble in a variety of solvents, including those in which it is coupled to the peptide or peptoid moiety and those in which it is administered to a subject. Those of ordinary skill in the art will recognize what is desirable solubility depending on the conditions in which the photosensitizer is coupled to form a conjugate (or the conditions in which the conjugate is administered). In an embodiment, the photosensitizer and targeting moiety can be coupled in a reaction requiring solubility in DMSO, water, ethanol, or a mixture thereof (e.g., a 1:1 mixture of DMSO: H 2 O or 5% ethanol in water). Various photosensitizers are known and can be used in the practice of this invention.
Photosensitizers typically have chemical structures that include multiple conjugated rings that allow for light absorption and photoactivation. They differ in properties of light absorption and fluorescence, biodistribution, temporal uptake, and mechanisms of photoactivatable cytotoxicity. One of ordinary skill in the art will appreciate that photosensitizers can include, but are not limited to, hematoporphyrins, such as hematoporphyrin HC1 and hematoporphyrin esters (Dobson et al., Arch. OralBiol. 37:883-887 (1992)); dihematophorphyrin ester (Wilson et al., Oral Microbiol. Immunol. 8:182-187 (1993)); hematoporphyrin IX and its derivatives (Russell et al., Can. J. App. Spectros. 36:103-107, available from Porphyrin Products, Logan, Utah); 3,1-meso tetrakis (o- propionamidophenyl) porphyrin; hydroporphyrins such as chlorin, herein, and bacteriochlorin of the tetra (hydroxyphenyl) porphyrin series, and synthetic diporphyrins and dichlorins; o-substituted tetraphenyl porphyrins (picket fence porphyrins); chlorin e6 monoethylendiamine monamide (CMA; Goff et al. 70:474-480 (1994); available from Porphyrin Products, Logan, Utah); mono-1-aspartyl derivative of chlorin e6, and mono- and diaspartyl derivatives of chlorin e6; the hematoporphyrin mixture Photofrin LI (quardra Logic Technologies, Inc., Vancouver, BC, Canada); benzophorphyrin derivatives (BPD), including benzoporphyrin monoacid Ring A (BPD-MA), tetracyanoethylene adducts, dimethyl acetylene dicarboxylate adducts, Diels-Adler adducts, and monoacid ring "a" derivatives; a naphthalocyanine (Biolo, Photochem and Photobiol. 5959:362-365 (1995)); toluidine blue O (Wilson et al., Lasers in Medical Sci. 8:69-73 (1993)); aluminum sulfonated and disulfonated phthalocyanine ibid.; and phthalocyanines without metal substituents, and with varying other substituents; a tetrasulfated derivative; sulfonated aluminum naphthalocyanines; methylene blud (ibid.); nile blue; crystal violet; azure β chloride; and rose bengal (Wilson, Intl. Dent. J. 44:187-189 (1994)). Numerous photosensitizer entities are disclosed in Wilson et al., (Curr. Micro. 25:77-81 (1992)) and in Okamoto et al. (Lasers in Surg Med. 12:450-485 (1992)). Other potential photosensitizer can include but are not limited to, pheophorbides such as pyropheophorbide compounds, anthracenediones; anthrapyrazoles; aminoanthraquinone; phenoxazine dyes; phenothiazine derivatives; chalcogenapyrylium dyes including cationic selena- and tellura-pyrylium derivatives; verdins; purpurins including tin and zinc derivatives of octaethylpurpurin and etiopurpurin; benzonaphthoporphyrazines; cationic imminium salts; and tetracyclines.
In an embodiment, the invention discloses a phenyl-xanthenone-based photosensitizer having a structure represented by formula (A):
Figure imgf000019_0001
wherein Ri, R2, R3, ^, R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R6 is selected from the group consisting of hydroxyl and alkoxy, and R is a moiety conjugated to the photosensitizer. hi an embodiment, R comprises a polymer. In an embodiment, R comprises a peptide or peptoid comprising at least one amino acid or amino acid side chain residue. More preferably, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the photosensitizer is 2,4,5,7-Tetraiodo-6-methoxy-9-(2,3,4,5- tetrachloro-phenyl)-xanthen-3 -one-based photosensitizer having a structure represented by
Figure imgf000020_0001
an embodiment, the photosensitizer is 2,4,5,7-Tetraiodo-6-methoxy-9-phenyl- xanthen-3-one having a structure represented by (A2):
Figure imgf000020_0002
In an embodiment, the photosensitizer is a benzothiazolyl-dihydroquinoline-based photosensitizer having a structure represented by formula (B):
Figure imgf000020_0003
wherein R' is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, and R is a moiety conjugated to the photosensitizer. h an embodiment, R comprises a polymer. In an embodiment, R comprises a peptide or peptoid comprising at least one amino acid or amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof. h an embodiment, the photosensitizer is 4-(3-Methyl-benzothiazol-2-ylmethylene)- 1,4-dihydro-quinoline having a structure of (Bi):
Figure imgf000021_0001
In an embodiment, the photosensitizer is 4-[3-(3-Methyl-benzothiazol-2-yl)- allylidene]-l,4-dihydro-quinoline having a structure of (B2):
Figure imgf000021_0002
In an embodiment, invention discloses a pyridinylidene-benzothiazole-based photosensitizer having a structure represented by formula (C):
Figure imgf000021_0003
wherein R" is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, and R is a moiety conjugated to the photosensitizer. In an embodiment, R comprises a polymer. In an embodiment, R comprises a peptide or peptoid. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof. In an embodiment, the photosensitizer is 3-Methyl-2-[3-(lH-pyridin-4-ylidene)- propenylj-benzothiazole having a structure of (Ci):
Figure imgf000022_0001
Peptide and Peptoid: The photosensitizer of the present invention is conjugated to either a peptide or a peptoid (See FIG. 1 A) to form a phototoxic compound. The peptide or peptoid is covalently conjugated to a photosensitizer via the amide group on the amino acid backbone, an embodiment, the peptide or peptoid comprises between about 1 and about 500 amino acid units in length, and can be the natural L-enantiomer, or the unnatural D-enantiomer, or a D- and L-enantiomer mixture. In an embodiment, the peptide or peptoid length is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500 amino acid units in length. Preferably, the length is between about 1 and about 25 amino acid units.
In an embodiment, the peptide or peptoid comprises at least one side-chain residue selected from aromatic amino acids (i.e., Phe, Tyr, Trp). In an embodiment, the side-chain residue from the aromatic amino acids is located proximate to the photosensitizer, i.e., about zero, or about one, or about two, or about three, or about four, or about five, or about six, or about seven, or about eight amino acid units away from the photosensitizer on the peptide. In an embodiment, the peptide or peptoid comprises side-chain residues from Table 1.
Table 1 Pe tides/Pe toids for Phototoxic Com ounds
Figure imgf000023_0001
In an embodiment, at least one amino acid side-chain residue on the peptide or peptoid is substituted with a non-amino acid side-chain residue. In an embodiment, the non- amino acid side-chain residue is a aryl group. As used herein, the term "aryl" refers to an aromatic group which has at least one ring having a conjugated pi electron system and includes without limitation carbocyclic aryl, aralkyl, heterocyclic aryl, biaryl groups and heterocyclic biaryl etc. In an embodiment, the non-amino acid side-chain reside is an indole- based, or a phenol-based, or an imidazole-based group (See FIG. IB.). In an embodiment, the substitution occurs at the amino acid unit that is about 0 to about 4 units away from the photosensitizer. h an embodiment, the substitution occurs at the unit most proximate (0 unit away from the photosensitizer) to the photosensitizer. hi an embodiment, further substitutions of the side-chain residue can be introduced for purposes of increased solubility, decreased aggregation and altered extent of hydrophobicity. Phototoxic Compound and Mechanism:
Based on the above disclosure, an embodiment provides a phototoxic compound comprising a photosensitizer described herein that is conjugated to a peptide or peptoid described herein via covalent bond. In an embodiment, the phototoxic compound can be represented by formula (1):
Figure imgf000024_0001
wherein Rls R , R , R4, R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof. h an embodiment, the phototoxic compound can be represented by formula (1 A) or
(IB):
Figure imgf000024_0002
Figure imgf000025_0001
wherein Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (2):
Figure imgf000025_0002
wherein Rls R2, R3, R^ R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, hydroxyl, R6 is selected from the group consisting of hydroxyl and alkoxy, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, and X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
In an embodiment, the phototoxic compound can be represented by formula (2A) or (2B):
Figure imgf000026_0001
wherein X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB. hi an embodiment, the phototoxic compound can be represented by formula (3):
Figure imgf000026_0002
wherein R10 is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12, and Xi is apeptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof. In an embodiment, the phototoxic compound can be represented by formula (3 A) or
(3B):
Figure imgf000027_0001
wherein Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (4):
Figure imgf000027_0002
wherein R10 is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, and X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from FIG. IB.
In an embodiment, the phototoxic compound can be represented by formula (4A) or
(4B):
Figure imgf000028_0001
or
Figure imgf000028_0002
wherein X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from FIG. IB.
In an embodiment, the phototoxic compound can be represented by formula (5):
Figure imgf000028_0003
wherein Rπ is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12, and Xi is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phtotoxic compound can be represented by formula (5A):
Figure imgf000029_0001
wherein X1 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, h an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof.
In an embodiment, the phototoxic compound can be represented by formula (6):
Figure imgf000029_0002
wherein Rπ is either hydrogen or alkyl group, m is 1, or 2, or 3, or 4, n is 1, or 2, or 3, or 4, or 5, or 6 or 7, or 8, or 9, or 10, or 11, or 12, and X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue. In an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from Figure IB.
In an embodiment, the phototoxic compound can be represented by formula (6A):
Figure imgf000030_0001
wherein X2 is a peptide comprising at least one amino acid, or a peptoid comprising at least one amino acid side chain residue, hi an embodiment, the peptide or peptoid comprises a side-chain residue from an aromatic amino acid, or a derivative thereof; and Y is one of the groups from FIG. IB.
The phototoxic compounds of the present invention are stable at room temperature and soluble in water and polar non-aqueous medium. This water solubility indicates that the phototoxic compounds of the present are able to form a soluble formulation with a pharmaceutically acceptable carrier and functionally interact with various tissues or cells, which have aqueous environment. The stability indicates that these phototoxic compounds are able to maintain their activity for a long time upon photo-induced activation, making these phototoxic compounds a very efficient agent for disease treatments. Furthermore, in an embodiment, the phototoxic compounds exhibit low cytotoxicity because they will not cause or contribute to an adverse biological reaction inside a tissue or cell due to their water solubility.
In an embodiment, the phototoxic compounds induce cytotoxic activity in a cell upon irradiation with light of proper energy and wavelength, hi an embodiment, the phototoxic compound acts in the cell via a mechanism of DNA cleavage. In an embodiment, the photosensitizer on the phototoxic compound absorbs light energy to cause a promotion of the photosensitizer from its ground state to the extremely unstable excited singlet state with a half-life in range of 10"6 to 10"9 seconds. The singlet excited photosensitizer either decays back to the ground state, resulting in the fluorescence or undergoes intersystem crossover to the longer lived (10"3 second) tripled excited state. The triplet photosensitizer reacts with ground state oxygen to produce singlet oxygen ( O2) while the triplet photosensitizer turns back to its ground state. The singlet oxygen then reacts with different biomolecules to cause oxidative damages. In particular, the singlet oxygen reacts with a side-chain residue on the conjugated peptide or peptoid moiety to form a highly active peroxide on the side-chain residue (See Scheme 1 (FIG. IC).) FIG. IC shows Scheme 1 which is a mechanism for DNA cleavage and phototoxicity of dye (S) - peptide conjugates. In an embodiment, the highly active peroxide then serves as scissors to directly cleave DNA and to prevent the DNA replication.
The phototoxic compounds possesses several advantages over those found in the prior art. The advantages include low toxicity, high stability and targeted cleavage of DNA, etc. The singlet oxygen produced by the prior art photosensitizers directly oxidizes a substrate in a cell. The substrate can be different types of proteins, or lipids, or RNAs, or DNAs. For example, Goosey et al. reported that a direct reaction between a singlet oxygen and a susceptible proteins results in non-disulfide covalent cross-links in the proteins. See Goosey et al., "Cross-linking of lens crystallins in a photodynamic system: a process mediated by singlet oxygen," Science, 208(4449): 1278-80 (June 13, 1980). See also U.S. Patent No. 6,607,522. Geiger et al. reported that cellular membranes, including plasma, mitochondrial and sometimes nuclear membranes, were severely damaged by oxidation of unsaturated fatty acid residues and of cholesterol. See Geiger et al., Photochem Photobiol, 62:580-587 (1995). In the case of oxidation of DNA, the prior art has taught that singlet oxygen directly reacted with DNA generating base damage that requires alkaline or heat workup for strand scission.
Figure imgf000031_0001
Scheme 1 : Mechanism for DNA cleavage and phototoxicity of a phototoxic compound The cleavage of DNA in a cell results in a cytotoxic effect on the cell. Preferably, two distinct modes of cytotoxic effect occur: necrosis and apoptosis. Necrosis refers to cell death in living tissues characterized by breakdown of cell membranes. Necrosis is always pathological. In necrosis, death of a large number of cells in one tissue area occurs as opposed to a method of selective cell death (apoptosis). Apoptosis is also known as programmed cell death. It can occur in normal tissues, for example, as a means of regulating cell numbers, and during embryological development. It is also seen in pathological processes. Apoptosis is brought about by a complex system of cell signaling pathways and enzyme events. Morphologically, apoptic cells shrink and the nucleus condenses. The organelles and the nucleus break up and the cells break into fragments. The light source for irradiation of the photosensitizers depends on the type of the tissue or cell and the location of the tissue or cell in a subject. In an embodiment, the light is from ultraviolet to visible and infrared red light. In an embodiment, the light is visible light. In an embodiment, laser light source can be used to provide the exact selection of wavelengths and the precise application of light. In an embodiment, the laser light sources include, but is not limited to, pulsed lasers such as the gold vapor laser and the copper vapor laser-pumped dye laser, tunable solid-state lasers such as the neodymium: YAG laser, and the semiconductor diode lasers etc.
The phototoxic efficiency with respect to oxidation of a target molecule by a phototoxic compound can be determined by both in vitro and in vivo assays well known in the art. For example, an in vitro assay can include measuring the ability of the phototoxic compound to "bleach" the substrate. An in vivo assay with a cell can include evaluating DNA degradation in the cell or apoptosis of the cell in the presence of the irradiated and unirradiated phototoxic compound, hi an embodiment, the apoptosis can be determined by measuring survival of the cell, hi an embodiment, the apoptosis can be determined by measuring DNA synthesis.
II. Synthesis of the Phototoxic Compound h an embodiment, the invention discloses a method of synthesizing a phototoxic compound. In an embodiment, the phototoxic compounds can be formed by a general process of synthesizing the photosensitizer part and the peptide or peptoid parts, followed by conjugating the photosensitizer parts to the peptide or peptoid parts.
Different approaches including those well known in the art, can be used to synthesize the photosensitizer parts. Examples of synthesizing a photosensitizer include but are not limited to teachings and disclosures in Neckers, D. C; Paczkowski, J. Tetrahedron, 42, 4671 (1986), Svanvik, N.; Westman, G.; Wang, D.; Kubista, M. Anal. Biochem., 281, 26 (2000), and Benson, S. C; Singh, P.; Glazer, A. Nucl. Acids Res., 21, 5727 (1993), the disclosures are herein incorporated by reference. Working examples 1 and 2 provide examples for the synthesis of a particular photosensitizer part.
Peptides can be synthesized by different methods well known in the art, including ribosomally-directed fermentation methods, as well as non- ribosomal strategies and chemical synthesis methods, h an embodiment, peptides containing the 20 natural amino acids and those greater than about 30 residues can be prepared via recombinant expression systems that utilize the ribosomally directed peptide synthesis machinery of a host organism, e.g., E. coli. In an embodiment, smaller peptides (less than 30 residues) and peptides which contain unnatural or non-proteinogenic amino acids or modified amino acid side chains often prepared through a more general solution-phase chemical synthesis of peptides (e.g., using N- Boc protection and the activated ester route). Protocols for sequence solution-phase chemical synthesis of peptides have been described in Andersson et al., Biopolymers 55:227-250 (2000). One current method used for generating peptides is solution-phase chemical synthesis, which employs a N-tert-butoxy (N-Boc) protected amino acid and a C-protected amino acid (Andersson et al., Biopolymers 55: 227-250 (2000)). An alternative solution- phase method for chemically-catalyzed peptide synthesis employs pre-activated esters as the carboxyl component for coupling (Andersson et al., Biopolymers 55: 227-250 (2000)). In addition, enzyme-mediated solid-phase peptide synthesis has also been employed. Solid- phase peptide synthesis (SPPS) uses insoluble resin supports, and has simplified and accelerated peptide synthesis and facilitated purification (Merrifield, R.B., J. Am. Chem. Soc. 85: 2149-2154 (1963)). Since the growing peptide is anchored on an insoluble resin, unreacted soluble reagents can be removed by simple filtration or washing without manipulative losses. In an embodiment, solid phase peptide synthesis can be performed using automation. A peptoid can be synthesized by a similar method to the synthesis of a peptide described above. For example, synthesis of a peptoid can be carried out by methods described in Murphy, J. E.; Uno, T.; Hamer, J. D.; Dwarki, V. Zuckermann, R. N., Proc. Natl. Acad. Sci. USA, 95, 1517 (1998), Simon, R. J.; Kania, R. S.; Zuckermann, R. N.; Huebner, V. D.; Jewell, D. A.; Banville, S.; Ng, S.; Wang, L.; Rosenberg, S.; Marlowe, C. K.; Proc. Natl. Acad. Sci. USA, 89, 9367 (1992), and Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J. Am. Chem. Soc, 114, 10646 (1992), all are incorporated herein by reference. In an embodiment, a photosensitizer-peptide conjugates (i.e., phototoxic compound) can be synthesized with a method known in the art. In an embodiment, the photosensitizer bearing an electrophilic moiety reacts with a nucleophilic group, i.e., amino terminus on a peptide or peptoid. For example the method can use commercially available Rink amide resin on a solid support for coupling the photosensitizer to the peptide or peptoid. Yield from coupling reactions can be assessed by spectroscopy. In an embodiment, couplings can be performed using 4 equivalents of Fmoc protected amino acid, 4 equivalents of HBTU and 8 equivalents of Hunig's base in DMF for 3 hours. In an embodiment, deprotection of the Fmoc group can be achieved using 20% piperidine in DMF for 30 minutes (to minimize diketopiperazine formation, dipeptides were deprotected using 50% piperidine in DMF for 5 min). In an embodiment, the dye moiety is attached to a resin-bound peptide as described below. In an embodiment, the dye-peptide conjugates are simultaneously deprotected and cleaved from the resin with a 95:5 TFA:ΗPS solution. The solution is then concentrated in vacuo and purified via RP-HPLC (H2O/CH3CN in 0.1% TFA). The resulting products can be isolated by lyophilization and characterized by MALDI-TOF mass spectrometry. For more examples of the phototoxic compound synthesis, one is referred to working examples 1 and 2.
III. Pharmaceutical Composition and Administration
In an embodiment, a pharmaceutical composition is disclosed that comprises a phototoxic compound described herein and a pharmaceutically acceptable carrier. Either solid or liquid pharmaceutically acceptable carriers can be employed. Solid carriers include but are not limited to, starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Liquid carriers include but are not limited to, syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like. Similarly, the carrier or diluent may include any prolonged release material. When a liquid carriers are used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension. A summary of such pharmaceutical compositions may be found, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton Pa. (Gennaro 18th ed. 1990). The pharmaceutical composition can be used in its solid form or dissolved in an appropriate solvent for addition to the carrier (solid or liquefied) or dissolved in an appropriate solvent. Mixtures should be in appropriate solvents for dissolving both medicament and carrier, and at the desired degree of medicament purity. In an embodiment, upon hydration, at the appropriate pH for the pharmaceutical composition, the phototoxic compound and the carrier form a complex which facilitates delivery of the phototoxic compound to a target. In an embodiment, other additives and pharmaceutical excipients can also be added, during or after formulation, to improve the ease of formulation, formulation stability, speed of reconstitution, delivery of the formulation. These include, but are not limited to, penetration enhancers, targeting aids, anti-oxidants, preservatives, buffers, stabilizers, solid support materials. In an embodiment, the composition may include osmoregulators if required, such as but not limited to, physiologically buffered saline (PBS), carbohydrate solution such as lactose, trehalose, higher polysaccharides, or other mjectable material. A wide variety of excipients and stabilizers are known in the art and their use will depend on formulation type and application requirements. The function of stabilizers is to provide increased storage stability in cases where the phototoxic compound or carrier is labile to heat, cold, light or oxidants or other physical or chemical agents. Other purposes for stabilizers can be for maintaining phototoxic compound and/or carrier in a form appropriate for transport to and uptake at the target site. Depending on the solubility, the excipients or stabilizers can be added either prior to deposition step or after the hydration step. In an embodiment, the pharmaceutical composition can be formulated into dosage forms such as capsules, impregnated wafers, ointments, lotions, inhalers, nebulizers, tablets, or mjectable preparations.
The pharmaceutically composition can be used to treat a variety of diseases or abnormal conditions. Non-limiting examples of disease include but are not limited to malignancies and inflammatory diseases, such as multiple types of cancer or tumor at different tissues and organs, demiatological diseases, atherosclerosis, infectious diseases, theumatoid arthritis, age-related macular degeneration, restenosis, ALDS, hematological diseases, etc.
The pharmaceutical composition can be administered by a variety of methods known in the art. One of ordinary skill in the art will appreciate that the route and/or mode of administration will vary depending on the conditions of target organisms and the desired results. For example, the pharmaceutical composition can be administered by methods including but being not limited to, oral, topical, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Effective amounts or doses of the composition for treating a disease or condition can be determined using recognized in vitro systems or in vivo animal models for the particular disease or condition. One of the factors that determine the dosage is the irradiation time. If it is desired to irradiate only for short time, the concentration of the composition can be increased. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dosage can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
In the case of cancer, many art-recognized models are known and are representative of a broad spectrum of human tumors. In an embodiment, the composition can be tested for inhibition of tumor cell growth in cell culture using standard assays with any of a multitude of tumor cell lines of human or nonhuman animal origin. Many of these approaches, including animal models, are described in detail in Geran, R. I. et al., "Protocols for Screening Chemical Agents and Natural Products Against Animal Tumors and Other Biological Systems (Third Edition)," Cane. Chemother. Reports, Part 3, 3:1-112. For example, a typical single dosage for administration to cells comprises between about 1 ng and about 500 mg of the active phototoxic compound per 1 x 105 cells, preferably between about 100 ng and about 100 mg of the active phototoxic compound per 1 x 105 cells. The dosage for administration to a subject comprises between about 1 ng and about 10 g of the active phototoxic compound per kilogram body weight, preferably between about 0.1 mg and about 100 mg per kilogram body weight.
Methods for irradiation include, but are not limited to, the administration of laser, nonlaser, or broad band light. Irradiation can be produced by extracorporeal or intraarticular generation of light of the appropriate wavelength, hi an embodiment, light used in the invention can be administered using any device capable of delivering the requisite power of light including, but not limited to, fiber optic instruments, arthroscopic instruments, or instruments that provide transillumination. Delivery of the light to a recessed, or otherwise inaccessible physiological location can be facilitated by flexible fiber optics (implicit in this statement is the idea that one can irradiate either a broad field, such as the lung or a lobe of the lung, or a nan-ow field where bacterial cells may have localized). Examples
The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, pending patent applications and published patents, cited through this application, are hereby expressly incorporated by reference.
Example 1. Preparation of rose bengal 5-carboxyl pentyyl ester-peptide conjugates (formula (1). (1 A). ( B), (2). (2A), and (2B))
The synthesis of formula (A) (precursor for formula (1) and (2)) was performed by the following method. Rose bengal 5-carboxy pentyl ester was synthesized according to the method of Neckers (Neckers, D. C; Paczkowski, J. Tetrahedron, 42, 4671 (1986)) by alkylation of one equivalent of rose bengal with 3 equivalents of 6-bromohexanoic acid in refluxing 1:1 H2O : Me2CO for about 24 hours. The crude material was purified via silica gel chromatography to give a red solid.
The synthesis of formula (1) was performed by the following method. The rose bengal moiety (RB) was attached by treating a resin-bound peptide (1.0 equiv.) with rose bengal 5-carboxy pentyl ester (3.0 equiv.), HBTU (3.0 equiv.), HOBt (3.0 equiv.) and DIPEA (6.0 equiv.) in DMF for about 12 hours.
The synthesis of formula (2) was performed by the following method. The erythrosin moiety (ER) was attached via an aminohexanoic acid spacer by treating a resin-bound peptide (1.0 equiv.) with erythrosin B isothiocyanate isomer II (1.0 equiv) and DIPEA (5 equiv.) in DMF for about 12 hours.
Example 2. Preparation of TO- and TO derivative-conjugates (formula (3). (3A\ (3B , (4). (4A (4B). (5\ (5 A . (6). and (6A))
The synthesis of formula (B) and (C) (precursors for formula (3), (4), (5) and (6)) was performed by the following method. Two derivatives of TO suitable for peptide coupling were prepared by Scheme 2 (FIG. 4B). The procedure followed literature methods with only slight modifications. See Svanvik, N.; Westman, G.; Wang, D.; Kubista, M. Anal. Biochem., 281, 26 (2000), and Benson, S. C; Singh, P.; Glazer, A. Nucl. Acids Res., 21, 5727 (1993). One derivative featured a carboxylate-terminated tether attached to the quinoline nitrogen (TOQ, 3), and the other featured the same linker attached to the benzothiazole nitrogen (TOz, 6). Derivatives of quinoline and benzothiazole were modified with 11 -bromoundecanoic acid (selected to minimize cyclization reactions that occurred with shorter linkers for TOz) and subsequently coupled with the appropriate heterocyclic quaternary salt to yield the cyanine dye containing a carboxylate functionality.
The synthesis of formula (3), (3 A), (3B), (4), (4A), (4B), (5), (5 A), (6), and (6A) was performed by the following method. Derivatives of TO suitable for peptide coupling were prepared by Scheme 3 (FIG. 4C). Quaternized nitrogen heterocyclic derivatives 7-8 were synthesized by carboxyalkylation of one equivalent 4-methylquinoline or 4-methylpicoline with one equivalent of 6-bromoundecanoic acid at about 110 °C for about 4 h. Derivative 9 was synthesized by alkylation of one equivalent of 2-methylbenzothiazole with three equivalents of iodomethane in refluxing dioxane and subsequently reacted with conjugated NN'-diphenylformamidine in refluxing acetic anhydride to give acetanilide derivative 9 which was coupled to either derivative 7 or 8 to yield the trimethine cyanine dye containing a carboxylate functionality. The thiazole orange moiety (TO) was attached by treating a resin- bound peptide (1.0 equiv.) with thiazole orange derivative (4.0 equiv.), HBTU (4.0 equiv.), and DIPEA (8.0 equiv.) in DMF for about 3 hours.
Example 3. Synthesis of a peptoid
Peptoids were synthesized according to the method of Zuckerman.4"6 The Fmoc-Rink amide resin (1.0 equiv.) was treated with 20% piperidine in DMF for about 30 minutes. The free resin-bound amine was then treated with a solution of bromoacetic acid (10 equiv.) and diisopropylcarbodiimide (10 equiv.) in DMF for about 30 minutes. This procedure was repeated. The resin was then treated with a solution of primary amine (40 equiv.) in DMF for about 12 hours. These two steps were repeated until an oligomer of desired length was obtained. The resin was then treated with Fmoc-protected amino acid (4.0 equiv.), HBTU (4.0 equiv.), and DIPEA (8.0 equiv.) in DMF for about 3 hours. The dye moiety was attached to the resin as described earlier. The dye-peptoid conjugates were simultaneously deprotected and cleaved from the resin with a 95:5 TFA : TIPS solution. The solution was concentrated in vacuo and purified via RP-HPLC (H2O/CH3CΝ in 0.1% TFA). The products were isolated by lyophilization and characterized by MALDI-TOF mass spectrometry. The purity of the peptides was > 95% as determined by analytical RP-HPLC (H2O/CH3CN in 0.1% TFA). Example 4. DNA-binding characterization of Thiazole Orange (TOVPeptide Conjugates
Materials and Methods:
Solvents were purchased from Fisher and reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Acros Organics (Morris Plains, NJ). Amino acids were purchased from Advanced ChemTech (Louisville, KY). Calf thymus DNA (CT DNA) was purchased from Sigma (St. Louis, MO). All solvents and reagents were used without further purification. HPLC grade acetonitrile and Millipore water were used for HPLC analysis. The buffer used in all experiments was 50 mM sodium phosphate, 10 mM sodium chloride (pH 7). Reversed-phase HPLC was performed using a HP 1100 system with a Varian 250 x
4.6 mm stainless steel column packed with Microsorb-MV 300 CI 8 (5 μM). A flow rate of about 1.0 mL/min. was used with an aqueous solution buffered with 50 mM ammonium acetate and a linear gradient from about 20 to about 100% acetonitrile over 80 min. HNMR spectra were recorded on a Varian 400 and 500 MHz spectrometer. Proton chemical shifts are reported in ppm (δ) relative to the solvent reference relative to tetramethylsilane (TMS) (de-DMSO, δ 2.50; CD3OD, δ 3.30). Data is reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m)], coupling constants [Hz], integration). Carbon NMR spectra were recorded on a Varian 500 (125 MHz) spectrometer with complete proton decoupling. Carbon chemical shifts were reported in ppm (δ) relative to TMS with the respective solvent resonance as the internal standard (DMSO, 39.5; CD3OD, 49.2). Mass spectral analysis was performed by the Boston College Mass Spectrometry Facility. Samples were analyzed by accurate mass electrospray mass spectrometry (ES-MS) operating in positive mode on a Micromass LCT mass spectrometer. UV analysis was performed on a Hewlett Packard 8452A Diode Array Spectrophotometer. Steady state fluorescence measurements were performed on a Jobin Yvon Horiba Fluorolog®-3. For all steady state measurements the
Figure imgf000039_0001
490- 650 run. Dissociation constants were measured using a Perkin Elmer Wallac Victor Fluorescence reader fitted with a 450-490 nm excitation filter and a 515 nm long pass emission filter. Hypochromicity measurements
Absorbance values for 4 μM solution of TO-conjugates were measured before and after the addition of 45 μM (bp) CT DNA. Percent hypochromicity was calculated as the percent change in absorbance at λmax.
Quantum yield measurements
Quantum yields are reported relative to a 50 nM fluorescein standard in 0.1 M NaOH (φ= 0.93). See Weber, G.; Teale, F. W. J. Trans. Faraday Soc. 53, 646 (1957). Absorbance values at the excitation wavelength (470 nm) were measured for solutions containing 13.5 μM TO-conjugate and 405 uM CT DNA bp. The sample was then diluted to a final TO- conjugate concentration of 1.5 μM and CT DNA concentration of 45 μM (bp) to ensure that the absorbance of the sample was less than 0.06 for measurement of emission spectra. The integral of the emission spectrum was corrected for variations in absorbance and reported relative to the fluorescein standard.
Measurement of dissociation constants Dissociation constants were determined with fluorescence titrations performed in a
384 well plate with a total volume of 40 μl in each well. The concentration of TO-conjugates was kept constant at 50 nM and the concentration of CT DNA was increased until fluorescence signals plateau. Each sample was run in triplicate and the values of each concentration point were averaged. Scatchard analysis was used to obtain Kd values (www.graphpad.com).
Diffusional quenching experiments
Samples containing 1.5 μM TO-conjugate and 45 μM CT DNA bp were titrated with hexaamineruthenium(ffl) chloride and the fluorescence was measured after each addition. The data was plotted according to the Stern- Volmer equation (Eq. 1) and the slope of the best-fit line was used to determine the value for kq. Lifetimes are relative to the reported lifetime for thiazole orange and were based on the ratio of the quantum yield of each sample to thiazole orange (φ= 0.11). See Netzel, T. L.; Nafisi, K.; Zhao, M.; Lenhard, J. R.; Johnson, I. J. Phys. Chem., 99, 17936 (1995), Nygren, T. L.; Svanvik, N.; Kubista, M. Biopolymers, 46, 39 (1998).
Figure imgf000041_0001
Displacement of distamycin A
The methods used for the displacement assay are analogous to those described by Boger. See Boger, D. L.; Tse, W. C; Bioorg. Med. Chem., 9, 2511 (2001), Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C; Hedrick, M. P. J. Am. Chem. Soc, 123, 5878 (2001). Fluorescence of a solution containing 2.0 μM TO-conjugate and 12 μM CT DNA bp was measured. Distamycin A was added to produce a final concentration of 2.0 μM and the fluorescence was measured once again. Percent decrease in fluorescence was calculated as the percent change in the integral of the emission spectrum upon addition of distamycin A.
General Protocol for the Synthesis of Peptides and TO-peptide Conjugate (FIG. 4):
TO-peptide conjugates were synthesized on solid support using commercially available Wang-Fmoc-Lys(Boc) (0.7 mmol/g, Advanced ChemTech). Couplings were performed using 4 equivalents of Fmoc protected amino acid, 4 equivalents of HBTU and 8 equivalents of diisopropylethylamine in DMF for about 3 hours. Deprotection of the Fmoc group was achieved using 20% piperidine in DMF for about 30 minutes (after coupling of the first amino acid to the resin, Fmoc deprotection was achieved using 50% piperidine in DMF for about 5 minutes in order to minimize diketopiperazine formation). The TO-peptide conjugates (TO) were simultaneously deprotected and cleaved from the resin with a solution of 9:1:1:1 trifluoracetic acid: triethylsilane: triisopropylethylamine: H2O for about 30 minutes. The solution was concentrated under reduced pressure in the presence of toluene in order to remove any residual TFA. TO-peptide conjugates were dissolved in a minimal amount of methanol, which was then removed under vacuum to yield a red solid. A more detailed synthesis protocol is described in Kelley et al., "Thiazole Orange-Peptide Conjugates: Sensitivity of DNA Binding to Chemical Structure," Organic Letters, 6(4): 517- 519 (2004, including the supporting information for this journal article.). The contents of this article is herein incorporated by reference in its entirety.
DNA-binding Characteristics of the Peptidointercalators:
All of the TO-peptide conjugates displayed the same DNA-dependent fluorescence as the parent compound (Table 2). Fluorescence quantum yields in the absence of DNA were very low (<0.0001), with about 80- to about 1300-fold increases in intensity observed upon the addition of calf thymus (CT) DNA (Table 1). TO is only emissive when the monomethine bridge connecting the two heterocyclic structures is rigidified through intercalation and exhibits a quantum yield of about 0.11 when DNA bound. Therefore, the observation of comparable quantum yields for most of the TO-peptide conjugates indicates that an intercalative binding mode is maintained.
Table 2. Photophysical Properties and DNA-Binding Affinities for TO-Peptide Conjugates compd Φrel (DNA) Kd (μM) Hypochιomicityc (%) Kq (X lO^M'V1) displacement (%)
TOQ-K 0.13 + 0.01 1.7 + 0.2 29 + 2 1.9 + 0.2 33 + 2
TOz-K 0.043 ± 0.001 0.9 ± 0.1 31 + 2 4.7 + 0.1 30 ± 5
TOQ-WK 0.078 ± 0.003 1.2 ± 0.3 26 ± 1 4.4 ± 0.5 40 + 6
TOz-WK 0.021 ± 0.002 3.1 ± 0.4 37 ± 2 12 ± 2 26 + 5
TOQ-GAQVWGFSAK 0.008 ± 0.002 not measured 9 ± 3 30 ± 9 34 ± 4
TOz-GAQVWGFSAK 0.012 ± 0.001 not measured 12 ± 1 38 ± 4 36 + 2
TOQ-KAQSWGKSAK 0.13 ± 0.01 0.4 ± 0.1 21 ± 1 1.4 ± 0.1 33 ± 2
TOz-KAQSWGKSAK 0.042 ± 0.002 0.5 ± 0.1 31 ± 2 3.4 ± 0.1 21 ± 1 Interestingly, most of the DNA-bound conjugates based on the TOQ derivative displayed higher quantum yields than those with the TOz scaffold. This trend may signify that the attachment of the peptide chain to the benzothiazole nitrogen impedes intercalation. Many intercalators bind from the minor groove of DNA. See Bloomfield, V. A.; Crothers, D. M.; Tinoco, I. Nucleic Acids: Structures, Properties, and Functions; University Science Books: Sausalito, CA, 2000. Therefore, the addition of substituents to a TO heteroatom that faces into the major groove might interfere with binding or alter the binding mode by requiring threading of the appendage through the DNA helix. The binding modes of DNA-bound TO-peptide derivatives were also monitored using a distamycin displacement assay (Table 2). See Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C; Hedrick, M. P. J. Am. Chem. Soc, 123, 5878 (2001). Distamycin binds to the minor groove of DNA and can block the binding of ligands that bind to DNA intercalatively, particularly if large functional groups reside in the minor groove. Interestingly, the binding of many of the TOQ derivatives was more sensitive to the presence of distamycin than the TOz counterparts. This trend may suggest that the TOz derivatives thread the attached peptide into the major groove, which could permit simultaneous binding of distamycin in the minor groove and the TOz-peptide conjugates. Example 5. Photosensitized DNA Cleavage Promoted by Amino Acids
Materials and Methods:
Solvents were purchased from Fisher and reagents were purchased from Aldrich Chemical Co. or Acros Organics. Amino acids were purchased from Advanced ChemTech. Both solvents and reagents were used without further purification. Reverse phase HPLC was performed on a Varian 250 x 4.6 mm stainless steel column packed with Microsorb-MV 300 C18 (5 mm). A flow rate of about 1.0 mL/min. was used with an aqueous solution buffered with 50 mM ammonium acetate and a linear gradient from 20 to 100% acetonitrile over 80 min. ιH NMR spectra were recorded on a Varian 400 MHz spectrometer. Proton chemical shifts are reported in ppm (d) relative to the solvent reference relative to tetramethylsilane (TMS) (dδ-DMSO, d 2.50). Data are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m)], coupling constants [Hz], integration). Carbon NMR spectra were recorded on a Varian 500 (125 MHz) spectrometer with complete proton decoupling. Carbon chemical shifts were reported in ppm (d) relative to TMS with the respective solvent resonance as the internal standard (DMSO, 39.5). Mass spectral analysis was performed by the Boston College Mass Spectrometry Facility. Samples were analyzed by accurate mass electrospray mass spectrometry (ES-MS) operating in positive mode on a Micromass LCT mass spectrometer. Absorbance spectroscopy was performed on a Hewlett Packard 8452A Diode Array Spectrophotometer.
DNA photocleavage experiments:
20 mM TO or TO-peptide conjugate was added to 75 mM (bp) pUC18 in 25 mM sodium phosphate (pH 7) in the dark [underivatized peptides] = 200 mM to ensure complexation with DNA). Irradiation was performed with a 365 nm transilluminating lamp. Cleavage efficiencies in aliquots removed from reactions were visualized after 1% agarose gel electrophoresis by ethidium staining. Minimal cleavage was observed when identical samples were incubated in the dark or when DNA samples were irradiated alone, hi FIG. 6B and FIG. 7A, percent cleaved values represent averages of 5-10 replicates, and a correction factor of 1.22 was used to adjust for the decreased stainability of supercoiled DNA. The data shown in FIG. 7A was obtained with 10 mM NaNs and 200 mM trolox. These scavenger concentrations produced minimal fluorescence quenching for DNA-bound TO-WK. FIG. 7A shows the effect of different agents on photocleavage of DNA by TO-WK; 5 minute irradiations were performed as described to quantitate changes in efficiency relative to TO-WK in buffered H2O under ambient conditions. FIG. 7B shows time dependence of Trp- based peroxide formation upon irradiation of TO-WK in 70% glycerol-30% X2O measured using a modified FOX assay.
Modified FOX assay:
The procedure for the modified FOX assay was based on that developed by Gebicki and co workers and described in ref. 15. 50 μM TO or TO-WK was added to a solution of 70% glycerol and 30% H2O or D2O in the dark. Samples were irradiated for 0, 15, 35, 45, or 60 minutes with a 365 nm transilluminating lamp. After irradiation, one volume of glacial acetic acid was added, followed by 700 μM xylenol orange and 280 μM ammonium iron(lT) sulfate hexahydrate. 50 μM H2O2 was added to a solution of 70% glycerol to provide a standard. Samples were thoroughly mixed after adding FOX reagents, and then incubated for 30 minutes in the dark. Samples were diluted with one volume of water, mixed, and the absorbance at 595 nm was measured using a Molecular Devices Thermomax microplate reader at 595 nm. The absorbance values for TO-WK were corrected for the small signals obtained with TO, and the activity of TO-WK was reported relative to that obtained with the H2O2 standard.
We describe a family of conjugates that derive DNA cleavage activity from a reaction that requires both a photoexcited intercalator and appended amino acids. FIG. 5 illustrates the series of compounds synthesized and tested for DNA photocleavage activity. The conjugates feature thiazole orange (TO), a fluorescent DNA intercalator,^ conjugated to synthetic dipeptides through a linker attached to the quinoline nitrogen of the heterocycle. These compounds were prepared using standard solid-phase peptide synthesis and a carboxy- functionalized TO derivative.- The first TO-dipeptide conjugates that were investigated and compared with the parent compound featured either glycine (TO-GK), tyrosine (TO-YK), or tryptophan (TO-WK) intervening between a terminal lysine and TO. These compounds display DNA-binding affinities and fluorescence quantum yields that are comparable to the parent compound.- Upon irradiation with visible light, the TO-WK conjugate efficiently cleaved supercoiled plasmid DNA (FIG. 6A), while TO-GK or underivatized TO did not produce significant levels of cleavage. FIG. 6 A shows photocleavage of pUC18 plasmid DNA by TO dipeptide derivatives analyzed by agarose gel electrophoresis. Solutions contained 20 μM TO or TO-peptide conjugate, 75 μM (bp) pUC18, and 25 mM sodium phosphate (pH 7). FIG. 6B shows time dependence of photocleavage activity for TO, TO-dipeptides and a WK dipeptide. Irradiation of DNA in the presence of a high concentration of a WK dipeptide or TO did not result in strand scission (FIG. 6B). These experiments indicated that the reaction observed required both TO and W. The involvement of both the intercalator and amino acid was confirmed by monitoring photocleavage in samples where TO and a W-containing peptide was introduced to plasmid DNA in trans.1 DNA photocleavage was detected even in the absence of a covalent linkage between TO and W, indicating that only the presence of these two reactants and light was required for the chemistry to occur.
A TO-YK conjugate also produced DNA cleavage upon photoexcitation, although with lower efficiency than TO-WK (FIG. 6B). The observation of activity for TO-WK and TO-YK that was significantly higher than for TO-GK indicates that the aromatic amino acids form reactive species in the presence of the TO excited state that are not accessible with the aliphatic amino acid.
To obtain information about the origin of the amino acid dependent DNA cleavage activity, a series of experiments was conducted to test for the involvement of diffusible species generated during photoexcitation of TO (FIG. 7A). Photocleavage of plasmid DNA by TO-WK was monitored in the presence of superoxide dismutase (SOD), catalase, and mannitol to test for the involvement of superoxide or hydroxyl radicals. The addition of these agents did not significantly affect the cleavage efficiency."
To determine whether photogenerated singlet oxygen contributed to the DNA cleavage reaction, the effect of D O, NaN , and argon was investigated (FIG. 7A). The introduction of D2O, a solvent that increases the lifetime of singlet oxygen,- increased the cleavage efficiency by over 50%. NaN3, a singlet oxygen scavenger,— decreased the cleavage efficiency by >65%. In addition, saturation of samples with argon before irradiation decreased the cleavage efficiency by >90%. These results strongly suggested that singlet oxygen was involved in the DNA cleavage reaction. Several features of the TO-peptide reactivity indicated that the cleavage mechanism was not a result of a direct reaction between JO2 and DNA. Singlet oxygen reacts with DNA, but typically generates base damage that requires alkaline or heat workup for strand scission.— The cleavage that is observed with the TO-peptide conjugates appears to involve direct strand scission, as no workup is required. Moreover, since the fluorophore responsible for generating 'θ2 is identical among the active and inactive TO-peptide conjugates, additional chemistry subsequent to the generation of O2 must occur to impart DNA photocleavage activity to TO-WK and TO-YK but not TO-GK.
A subset of naturally-occurring amino acids is known to react with singlet oxygen to form peroxides.— Tip and Tyr efficiently react with 1O2, with quenching rate constants (kiot) of 3.2 x 107 and 0.5 x 107 M^s-1, respectively.— Gly exhibits very low reactivity upon exposure to ^2, with ktot < 0.1 x 107 M^s-1. The trend in the rate constants coincides with the DNA cleavage activity of the TO-peptide conjugates.
As shown in FIG. 7A, trolox, a peroxyl-radical scavenger,— significantly decreased the efficiency of DNA photocleavage by TO-WK (Figure 7A). To test directly whether peroxides were formed upon irradiation of the TO-peptide conjugates, a modified FOX assay was employed.— This analysis showed significant levels of peroxide formation upon irradiation of TO-WK that increased when D2O was introduced into the samples. These results are consistent with the production of amino-acid based peroxides formed by a reaction with O2 generated by photoexcited TO. A previous report of DNA cleavage by thermally- generated peroxides provides a precedent for strand scission by this class of chemical species.1^
The DNA-binding peptide-intercalator conjugates described here exhibit DNA cleavage activity that appears to result from the reaction of !O with amino acids. Damage to protein side chains is proposed to be a potential source of the deleterious effects of O2 — The discovery of a model system that permits the photogeneration of !O in proximity to reactive residues will facilitate studies of this damage pathway. Moreover, TO-peptide conjugates will provide useful tools for analysis of the chemical reactions of amino acid- based peroxides with DNA.
Notes and references
1 T. Le Doan, L. Perrouault, D. Praseuth, N. Habhoub, J. L. Decout, N. T. Thuong, J. Lhomme and C. Helene, Nucleic Acid. Res., 1987, 15, 7749; P. E. Nielsen, C. Jeppesen, M. Egholm and O. Buchardt, Biochemistry, 1988, 27, 6338; A. Sitlani, E. C. Long, A. M. Pyle and J. K. Barton, J. Am. Chem. Soc, 1992, 114, 2303; B. Armitage, T. Koch, H. Frydenlund, H. Orum, H. G. Batz and G. B. Schuster, Nucleic Acid. Res., 1997, 25, 4674; H. Yu, J. C. Quada, D. Boturyn and S. M. Hecht, Bioorg. Med. Chem., 2001, 9, 2303; P. Fu, P. M. Bradley, D. van Loyen, H. Durr, S. H. Bossmann and C. Turro, Inorg. Chem., 2002, 41, 3808.
2 C. H. Tung, Z. Wei, M. J. Leibowitz and S. Stein, Proc Natl. Acad. Sci., USA, 1992, 89, 7114; M. P. Fitzsimons and J. K. Barton, J. Am. Chem. Soc, 1997, 119, 3379.
3 M. Petersen and J. P. Jacobsen, Bioconjugate Chem., 1998, 9, 331.
4 J. Nygren, N. Svanvik and M. Kubista, Biopolymers, 1998, 46, 39.
5 See ESIf for description of the synthesis and characterization of TO- peptides and precursors.
6 The Kά values corresponding to TO, TO-GK, TO-WK, and TO-YK bound to calf thymus (CT) DNA were 1.8 ± 0.1, 2.1 ± 0.1, 2.4 ± 0.3, and 2.9 ± 0.2 μM, respectively. The quantum yields of TO, TO-GK, TO- WK, and TO-YK bound to CT DNA were 0.11,4 0.20 ± 0.02, 0.16 ± 0.02, 0.22 ± 0.01. See ESIf for procedures and conditions used to obtain these values.
7 Solutions containing 20 μM TO, 200 μM KWK, and 75 μM bp pUC18 DNA exhibited cleavage yields that were ~ 50% relative to the covalent TO-WK conjugate. No cleavage was observed with TO + DNA or KWK + DNA after irradiation.
8 In the presence of 100 mM D-mannitol, 5 ng ul2l SOD, 5 ng ul21 catalase, or 5 ng ul2l SOD and 5 ng ul21 catalase cleavage yields did not vary more than 15%.
9 P. B. Merkel, R. Nilsson and D. R. Kearns, J. Am. Chem. Soc, 1972, 94, 1030.
IO C S. Foote, T. T. Fujimoto and Y. C. Chang, Tetrahedron Lett, 1972, 45; N. Hasty, P. Merkel, P. Radlick and D. R. Kearns, Tetrahedron Lett, 1972, 49.
11 A. W. M. Nieuwint, J. M. Aubry, F. Arwert, H. Kortbeek, S. Herzberg and H. Joenje, Free Radical Res. Commun., 1985, 1, 1.
12 A. Wright, W. A. Bubb, C. L. Hawkins and M. J. Davies, Photochem. Photobiol, 2002, 76, 35; I. Saito, T. Matsuura, M. Nakagawa and T. Hino, Ace Chem. Res., 1977, 10, 346. 13 A. Michaeli and J. Feitelson, Photochem. Photobiol, 1994, 59, 284.
14 F. Regoli and G. W. Winston, Toxicol. Appl. Pharmacol, 1999, 156, 96.
15 C. Gay, J. Collins and J. M. Gebicki, Anal. Biochem., 1999, 273, 149-155.
16 T. Paul, M. J. Young, I. E. Hill and K. U. igold, Biochemistry, 2000, 339, 4129.
17 P. E. Morgan, R. T. Dean and M. J. Davies, Eur. J. Biochem., 2002, 269, 1916.
Example 6. Phototoxicity of peptide conjugates
HeLa cells were incubated with 10 μM TO-peptide conjugates (phototoxic compounds) as shown in FIG. 3. The HeLa cells together with the phototoxic compounds were irradiated with visible light for about 10 minutes and incubated for about 24 hours before counting the cells for apoptosis or survival rate. Fluorescence imaging of HeLa cells incubated with TO-peptide conjugates demonstrated cellular uptake of the phototoxic compound (See FIG. 2). FIG. 2 shows fluorescence imaging of HeLa cells incubated with TO-tat conjugate demonstrating cellular uptake.
The compounds such as TO-WK and TO-WRKKRRQRRR formed peroxides (See FIG. 7B) and exhibited significant and selective phototoxicity.
Example 7. Phototoxicity of Peptidoconjugates Modulated by a Single Amino Acid
Oxidative stress resulting from the intracellular release of chemical oxidants or free radicals is known to exert deleterious effects on biological function. In cells, exposure to light or sensitizers can produce singlet oxygen (!O2), a highly reactive, mutagenic, and genotoxic species that induces oxidative stress. These properties have been harnessed in photodynamic chemotherapy, an anticancer treatment that involves the photosensitization of ]O2 to promote cell death within solid tumors. Chemical modification of essential cellular components likely underlies the detrimental effects of ^2, as direct damage to DNA, proteins and lipids is observed. Given that the reaction of 102 with DNA and amino acids generates transient species that are highly reactive (e.g. endoperoxides and peroxyl radicals), cross- reactions between nucleic acids and bound proteins are probable. Understanding and exploiting biomolecular cross-reactions that occur when O2 is generated intracellularly may lead to new photodynamic therapeutics with enhanced properties. Described herein is a strategy toward the study of oxidative cross-reactions promoted by ^2 between amino acids and DNA that relies on a family of DNA-binding peptidoconjugates bearing the photoactive intercalator thiazole orange (TO). Upon photoexcitation, TO generates xOι, thus serving as an oxidant source and providing a DNA- binding anchor. Certain TO-dipeptide conjugates exhibit DNA-photocleavage activity which depends on the composition of the peptide. Conjugates that contain certain aromatic amino acids, particularly tryptophan (W) and tyrosine (Y), are capable of DNA photocleavage, whereas conjugates containing glycine (G) or phenylalanine (F) do not promote strand scission. An explanation for such DNA-cleavage activity is an identified subset of protein residues (including tryptophan, cysteine, histidine, and tyrosine) which react with 2O2 to form peroxides. Peroxyl radicals are reasonable candidates as the active DNA-cleaving species, as precedent exists for strand scission through hydrogen abstraction from the DNA backbone by thermally-generated peroxides. Indeed, studies of TO peptidoconjugates displaying strand- scission activity revealed that amino acid based peroxides are the active species that induce DNA cleavage.
Discussed herein are TO peptidoconjugates that access human cells and exhibit amino-acid-dependent phototoxicity. The TO conjugates feature a portion (residues 49-57) of the HIV-1 transactivator of transcription (Tat) peptide sequence, which was previously used by other research groups to deliver appended cargoes into cells, investigated herein is whether cell-permeable TO peptidoconjugates are toxic to human cells upon photoexcitation, and to determine if toxicity can be triggered by the presence of specific amino acids. Indeed, a TO-Tat peptidoconjugate containing tryptophan cleaves DNA in vitro and exhibits appreciable phototoxicity, wherea a glycine-containing analogue neither elicits significant DNA cleavage, nor causes cell death. These examples present an example of novel peptide- containing photodynamic agents with activities that can be tuned through the manipulation of sequence composition.
FIG. 9A and FIG. 9B show various compounds utilized in the present experiment. FIG. 9A shows structures of TO peptidoconjugates: compound la shows TO-W-Tat; compound 2a shows TO-GK; and compound 2b shows TO-WK. FIG. 9B shows photocleavage of pUCl 8 plasmid DNA by TO peptidoconjugates analyzed by agarose gel electrophoresis. Solutions contained pUC18 (75μM (base pairs)), sodium cacodylate (lOmM, pH 7), and TO peptidoconjugates (luM). Samples were irradiated (λ=501 nm) for 30 minutes as indicated (+or- hv), and the conversion of supercoiled to nicked circular plasmid was monitored to evaluate DNA cleavage.
As shown in FIG. 9A, TO-D-peptide conjugates la-2b were prepared on Rink amide solid support, and the N-terminus was capped with a TO derivative by using standard solid phase Fmoc chemistry. Subsequent cleavage from the resin and purification by HPLC afforded TO peptidoconjugates la-2b. The non-natural D-peptide structure was used to impart resistance to proteolytic degradation. The two versions of the Tat peptide, la and lb, were prepared with glycine or tryptophan residues, respectively, positioned proximal to the dye to evaluate is selective DNA cleavage and phototoxicity could be observed. The TO- dipeptide conjugates 2a and 2b are analogues to L-amino acid containing conjugates and are used here as positive controls for DNA cleavage.
The photocleavage properties of the TO-Tat peptidoconjugates were investigated with a plasmid nicking assay. Upon irradiation with visible light, the tryptophan-containing conjugate lb caused high levels of strand scission as shown in FIG. 9B. The observation that lb yields greater DNA cleavage (induced by the tryptophan-based peroxide formed upon the production of !O2) than la (which elicits only low-level cleavage from direct 1O2-ρromoted damage) indicates that these TO conjugates exhibit photoreactivity analogous to that of their L-amino acid counterparts. Interestingly, 2b, a control dipeptide conjugate comprised of D- amino acids, cleaved supercoiled plasmid DNA more efficiently than lb, thus demonstrating that tryptophan exhibits greater reactivity when presented to DNA within the context of a dipeptide rather than the Tat decapeptide. This effect may arise from different tryptophan conformations that are induced by the two peptides. Nonetheless, these results indicate that the amino acid dependent DNA cleavage for dipeptide conjugates can be extrapolated to a more complex peptide structure. Furthermore, the TO-W-Tat peptidoconjugate, which should have good cellular uptake properties, is a suitable probe for studies inside cells.
Confocal fluorescence microscopy confirmed cellular uptake of the TO-Tat peptidoconjugates. As TO undergoes a dramatic increase in fluorescence quantum yield when bound to DNA or RNA, it can be used as an intrinsic probe for the location of the conjugates within cells. Both TO-Tat peptidoconjugates la and lb exhibited identical localization patterns, indicating they were efficiently imported into living unfixed HeLa cells as shown in FIG. 10A-FIG. IOC. FIG. 10A shows transmission image of cells incubated with compound lb (shown in FIG. 9A); FIG. 10B shows red-fluorescence image of the same cells that illustrates both cytoplasmic and nuclear uptake of lb; and FIG. IOC shows peptidoconjugate la shows an identical internalization pattern to that of lb. Less than five percent of the cells were stained when treated with propidium iodide, a dye specific for dead cells, thus reflecting that the conditions used to evaluate uptake patterns of the peptidoconjugates did not induce cell death.
In order to further investigate the location of the TO-Tat peptidoconjugates within human cells, colocahzation experiments were performed with two control dyes. SYTO-85 binds to nucleic acids and provides a marker for nuclear uptake, and mitotracker deep red- 633 selectively stains mitochondria. Interestingly, the TO-Tat peptidoconjugates were found to enter both the mitochondria (FIG. 11 A-FIG. 1 IC) and the nucleoli (FIG. 1 ID-FIG. 1 IF) of live cells. FIG. 11A-1 IF show localization profiles of TO-Tat peptidoconjugates. HeLa cells were incubated with la (10μM) and SYTO-85 (500nM) or mitotracker red-633 (3μM) for 1.5h at 37°C. FIG. llA and FIG. 1 ID show red-fluorescence image of cell stained with la. FIG. 11B shows visualization of mitotracker deep red-633 staining of the mitochondria. FIG. 11C shows merged image of red- and blue-fluorescence images illustrating colocahzation of la with mitotracker deep red-633. FIG. 1 IE shows green-fluorescence image illustrating nucleolar staining by SYTO-85. FIG. 1 IF shows merged image showing that la colocalizes with SYTO-85 in the nucleoli of these cells. Red fluorescence: λex=488 nm, λem=500-571nm; greenfluorescence: λex=:543nm, λem =556-648 nm; blue fluorescence: λex=543 nm, λem =600- 750 nm.
This finding is in stark contrast to previous work which showed that the Tat sequence did not enter the mitochondria. However, the peptide had been conjugated to a different dye (Oregon green); different cargoes can markedly change uptake and localization patterns. The TO conjugates described herein are most emissive when bound to DNA. Therefore, it is clear that the compounds have penetrated the nucleus, there may also be appreciable concentrations of the compounds in regions of the cell that appear dark in the images above.
As the TO-Tat peptidoconjugates were shown to enter human cells efficiently, these probes presented an appropriate system for the analysis of phototoxicity. When incubated with HeLa cells and irradiated, the TO-W-Tat peptidoconjugate lb was significantly phototoxic to cells as a function of irradiation time, whereas cells exposed to the glycine- containing conjugate la were unaffected as shown in FIG. 12. FIG. 12 shows the phototoxicity of TO-Tat peptidoconjugates. Toxicity was evaluated 12 hours after incubation (3μM) and irradiation (λ=501nm) with la (represented by circles) and lb (represented by squares) by CCK-8 assay. Data points show mean values; error bars show standard-deviation values. All assays were performed in triplicate, and three multiple, independent trials were conducted. Controls were performed in the dark to confirm that cell viability was maintained in the absence of photoexcitation. Light controls were also performed to confirm that the irradiation conditions did not harm the cells.
After irradiation, the cells were incubated with fresh media for 24h before viability was analyzed to allow the effects of the compounds to be assessed. Cell death was not observed if cells containing either conjugate were kept in the dark. Interestingly, the Tat peptide was observed to effectively abolish the dark toxicity of the parent TO compound, as unmodified TO causes quantitative cell death even in the absence of light.
The correlation of phototoxicity with the DNA-photocleavage activity of the TO peptidoconjugates described herein strongly suggests that the tryptophan-based peroxides fomied on TO conjugates containing this amino acid are responsible for this selectivity.
Whereas it cannot be unequivocally proven that the decreased cell viability results from DNA cleavage, the location of the conjugates within the cellular compartments that contain genomic DNA presents the possibility that strand scission may underlie the toxicity described. This represents the first study in which designed Tat peptide sequences were used to induce selective DNA damage and to produce phototoxic agents.
Experimental Section
Dye-peptide conjugates: Solid-phase synthesis was performed with Rink amide resin (NovaBiochem). Couplings were performed with Fmoc-protected D-amino acid (4 equiv, Advanced ChemTech, Fmoc=9-fluorenylmethyloxycarbonyl), HBTU (4 equiv, Advanced ChemTech, HBTU^O- enzotriazol-l-y -N^N^N'-tetramethyl-uronium hexafluorophosphate), and N,N-diisopropylethylamine (8 equiv, Acros) in N,N-dimethyl formamide (DMF) for 3h. The Fmoc group was removed with piperidine (20% v/v) in DMF for 30 min. (To minimize diketopiperazine formation, dipeptides were deprotected with piperidine (50% v/v) in DMF for 5 min). The deprotected N termini were capped with TO- COOH (4 equiv) under standard coupling conditions as described above. To minimize the formation of by-products resulting from Rink amide resin at high concentrations of trifluoroacetic acid (TFA), a two-step procedure for detachment/deprotection of the resin was performed as described. The dye-peptide conjugates were detached from the resin by slurrying in TFA/CH2CL2 (10% v/v) and transferred to a glass funnel with a fine sinter. The solvent was allowed to drip slowly through the resin bed and washed with TFA/CH2C12 (5% v/v) and concentrated in vacuo. Deprotection was carried out by stirring the residue in
TFA/TIS (95:5) at room temperature for 0.5-2h (TIS-triisopropylsilane, Acros). The solution was concentrated in vacuo and Et2O was added to precipitate the peptide. The resulting red solid was dissolved in TFA/H2O (0.1% v/v) and purified by reversed-phase HPLC (H2O/CH3CN in TFA (0.1% v/v)). The products were isolated by lyophilization and characterized by MALDI-TOF MS. The purity of the peptides was . 95% as determined by analytical reversed-phase HPLC (H2O/CH3CN in TFA (0.1 % v/v)). A molar extinction coefficient of ε=63000M"1cm"1 in H2O (λ=500nm) was used to quantify TO-peptide conjugates.
DNA photocleavage: TO-peptide conjugate (lμM) was added to pUC18 (75μM (base pairs)) in sodium cacodylate (lOmM, pH 7) in the dark. Irradiation was performed for 30 min (λ=501 nm) with an Oriel Instruments spectral luminator tunable light source. The lamp intensity was 1.36 mWcm-2. Cleavage efficiencies were evaluated by agarose gel (1%) electrophoresis visualized by ethidium bromide staining. Minimal cleavage was observed when identical samples were incubated in the dark, or when DNA samples were irradiated alone.
Cell culture: HeLa 229 cells (ATCC) were cultured as subconfluent monolayers on cell culture plates (25 or 75 cm2) with vent caps (Corning) in 1 X minimum essential α medium (Gibco) supplemented with fetal bovine serum (10% v/v, ATCC) in a humidified incubator at 37°C containing CO2 (5%). Confocal microscopy: HeLa cells that had been grown to subconfluence were dissociated from the surface with a solution of ethylenediaminetetraacetic acid (EDTA, 0.53mM)/trypsin (0.05%)(2mL, Cellgro) for 15 minutes at 37°C. Aliquots of 1X105 cells were plated in four-well Lab-Tek glass-bottom coverslips (Nalge Nunc) and cultured overnight to allow cell adherence. The culture medium was removed, and the cells were rinsed in IX Ca2+- and Mg2+- free phosphate-buffered saline (PBS, pH 7.4, Cellgro). HeLa cells were incubated for 1.5 h at 37°C with media (500μL) containing la or lb (as shown in FIG. 9A) (lOμM). For colocahzation studies, cells were incubated with la (lOμM) and SYTO-85 (500nM, Molecular Probes) or mitotracker deep red-633 (3μM, Molecular Probes). Cells were washed three times for 5 minutes with PBS (lmL). After washing, PBS (500μL) was added and the cells were placed on ice. Images were taken with an inverted Leica TCS SP2 scanning confocal microscope with an oil immersion lens (40x). The images were analyzed with the Leica confocal software program.
Cells incubated with SYTO-85, mitotracker deep red-633, or compound la (as shown in FIG. 9A) were used to identify appropriate emission-collection parameters and to minimize bleed through of the colocalized fluorophore. The excitation wavelength for both compound la and lb of FIG. 9A
Figure imgf000054_0001
was 488nm, and emission was collected in the range of 500-571nm. SYTO-85 (λexmax =567 nm) and mitotracker deep red-633 (λex maχ-644 nm) were excited at 543 nm, and emissions were collected in the ranges of 556-648 and 600-750 nm, respectively. These parameters were used in all experiments. Cells were exposed to propidium iodide (MP Biomedicals) to determine the extent of cell death (5%). Propidium iodide was excited at 488 nm, and emission was collected in the range of 550-700 nm. Cells that fluoresced brightly in subsequent experiments were assumed to be dead, and were not used in the evaluation of the conjugates.
Phototoxicity: HeLa cells were split as described above, and aliquots (lOOμL) were seeded (1X104 cells) into 96-well clear flat-bottom microplates (Costar). After overnight incubation, the medium was replaced with new media (lOOμL). Freshly prepared solutions of compounds la and lb (as shown in FIG. 9A) (3μM) were added to each well. Cells were incubated for 3 minutes in the dark at 37oC and then irradiated (λ=501nm) for 2, 5, 10, 15, and 20 minutes (UVA doses of 0.804, 2.01, 4.02, 6.03, and 8.04 Jem"2, respectively) in triplicate with an Oriel Instruments spectral luminator tunable light source. The cell medium was replaced after the entire plate was irradiated. For the dark controls, fresh medium was also added.
Cells were analyzed with the cell counting kit-8 (CCK-8, Dojindo) to determine cell viability. After overnight incubation following irradiation, the medium was removed and fresh medium (90μL) containing CCK-8 (lOμL) was introduced. After incubation at 37°C for lh, the absorbance of each sample was measured (λ=470 nm) on a thermamax plate reader (Molecular Devices). The data for samples that contained only CCK-8 and medium were subtracted from all samples. Wells without conjugate were used as controls to determine the extent of cell death. FIG. 12 represents an average of these three separate trials. Example 8. Differential phototoxicity of nuclear and mitochondrial oxidants
FIG. 8A-8C shows differential phototoxicity of nuclear and mitochondrial oxidants. FIG. 8 A shows a phototoxic compound is specific for a nucleus of a cell. FIG. 8A shows the localization of the nucleus specific peptidoconjugate. In an embodiment, the nucleus specific peptidoconjugate is TO-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg. FIG. 8B shows a phototoxic compound is specific for a mitochondria of a cell. FIG. 8B shows the localization of the mitochondria specific peptidoconjugate. In an embodiment, the mitochondria specific peptidoconjugate is TO-Phe-d-Arg-Phe-Lys. FIG. 8C shows a % survival of treating cells in a mitochondria. As shown, the mitochondria specific peptidoconjugate displays a greater ability to treat mitochondria of a cell. As such, the disclosed compounds allow for localized treatment of various conditions.
Other embodiments
All references disclosed herein are incorporated herein by reference. The present invention is not to be limited in scope by the specification embodiments described, which are intended as single illustrations of individual aspects of the invention. Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.

Claims

We claim:
A phototoxic compound of formula (1):
Figure imgf000056_0001
wherein : Rls R2, R3, R4, R5, R , R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
R6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and
Xi is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid.
2. The phototoxic compound of claim 1, wherein the amino acid side chain of X} is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
3. The phototoxic compound of claim 1 , wherein:
Rl5 R2, R3, R4 are each CI; n=5;
R5, R7, R8, and R9 are each I; and
R6 is MeO.
4. The phototoxic compound of claim 3, wherein the amino acid side chain of Xi is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
5. The phototoxic compound of claim 1 wherein:
Ri, R , R3, and are each hydrogen; n=5;
R5, R7, R8, and R9 are each I; and
R6 is MeO.
6. The phototoxic compound of claim 5, wherein the amino acid side chain of Xi is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
7. A phototoxic compound of formula (2) :
Figure imgf000057_0001
wherein:
Ri, R2, R3, i, R5, R7, R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
R6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; X is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid; and
Y is one selected from the group consisting of:
Figure imgf000058_0001
wherein als a2, a3, a4, a5, bi, b2, b3, and b4 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, and hydroxyl.
8. The phototoxic compound of claim 7, wherein the amino acid side chain of X2 is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
9. The phototoxic compound of claim 7, wherein:
Ri, R2, R3 and t are each CI;
R5, R7, R8 and R9 are each I;
n=5; and
R6 is MeO.
10. The phototoxic compound of claim 9, wherein the amino acid side chain of X2 is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, andYRKKRRQRRR.
11. The phototoxic compound of claim 7, wherein: Ri, R2, R , and R4 are each hydrogen; n=5;
R5, R7, R8, and R9 are each I; and R6 is MeO.
12. The phototoxic compound of claim 11 , wherein the amino acid side chain of X2 is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
13. A method of cleaving DNA in a sample comprising:
contacting a phototoxic compound of the formula:
Figure imgf000059_0001
wherein:
Ri, R2, R , Rj, R5, R , R8 and R9 are each independently selected from the group consisting of hydrogen, halogen, alkyl, and hydroxyl;
R6 is selected from the group consisting of hydroxyl and alkoxy; n is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12; and Xi is a peptide comprising at least one aromatic amino acid, or a peptoid comprising at least one amino acid side chain, wherein at least one amino acid side chain is from an aromatic amino acid;
with the sample; and
irradiating the sample and the phototoxic compound with visible light to activate the phototoxic compound.
14. The method of claim 13, wherein the amino acid side chain of Xi is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
15. The method of claim 13, wherein: Ri, R , R , Rt are each CI; n=5;
R5, R7, R8, and R9 are each I; and R6 is MeO.
16. The method of claim 15, wherein the amino acid side chain of Xi is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
17. The method of claim 13, wherein:
Ri, R2, R3, and j are each hydrogen; n=5;
R5, R7, R8, and R9 are each I; and
R6 is MeO.
18. The method of claim 17, wherein the amino acid side chain of Xi is selected from an amino acid side chain of the group consisting of K, WK, YK, GK, GAAQVWGFSAK, KAQSWGKSAK, GRKKRRQRRR, WRKKRRQRRR, HRKKRRQRRR, and YRKKRRQRRR.
19. The method of claim 13 further comprising transferring the phototoxic compound into a cell.
20. The method of claim 13 further comprising administering a pharmaceutical compound comprising the phototoxic compound to a patient.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021211969A3 (en) * 2020-04-17 2022-01-06 Idexx Laboratories, Inc. Fluorescence quenching immunossay
US11910795B2 (en) 2013-03-15 2024-02-27 Suncor Energy Inc. Natural indole auxin and aminopolycarboxylic acid herbicidal compositions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHILVERS ET AL: "Phototoxicity of rose bengal in mycological media-implication s for laboratory practice.", LETT APPL MICROBIOL., vol. 28, February 1999 (1999-02-01), pages 103 - 107, XP002992926 *
CONLON ET AL: "Site-directed photochemical disruption of the actin cytoskeleton by actin-binding Rose Bengal-conjugates.", J PHOTOCHEM PHOTOBIOL. B BIOLOGY., vol. 68, November 2002 (2002-11-01), pages 140 - 146 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11910795B2 (en) 2013-03-15 2024-02-27 Suncor Energy Inc. Natural indole auxin and aminopolycarboxylic acid herbicidal compositions
WO2021211969A3 (en) * 2020-04-17 2022-01-06 Idexx Laboratories, Inc. Fluorescence quenching immunossay

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