Patent Application
20240368229
The instant application contains a Sequence Listing which has been submitted in ASCII format via Patent Center and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 27, 2024, is named “047162-7170US2_Sequence Listing,” and is 705,600 bytes in size.
Cell-penetrating peptides as vectors for cell entry show great promise as research tools or for drug delivery. However, the majority of cell-penetrating peptides and thus the associated cargo molecules are sequestered in endosomes and eventually degraded.
There is a need in the art for cell-penetrating peptides that allow for transport of attached cargo into the cell, and yet avoid endosome sequestration and consequent degradation of the cargo. The present disclosure addresses this need.
In one aspect, the invention provides a method of delivering a cargo molecule to the cytoplasm of at least one cell, the method comprising contacting the cell with a transport construct comprising the cargo molecule and at least one cell-penetrating peptide selected from the group consisting of SEQ ID NOs:1-164, or a salt or solvate thereof.
In another aspect, the invention provides a method of promoting endosomal escape for a cargo molecule, the method comprising contacting at least one cell with a transport construct comprising the cargo molecule and at least one cell-penetrating peptide selected from the group consisting of SEQ ID NOs:1-164, or a salt or solvate thereof.
In various embodiments, the cell-penetrating peptide is SEQ ID NO: 2.
In various embodiments, the transport construct further comprises at least one selected from the group consisting of an activity modulating flanking sequence, a temperature modulating flanking sequence, and a pH modulating flanking sequence.
In various embodiments, the transport construct further comprises a linker connecting the cargo molecule and the cell-penetrating peptide.
In various embodiments, the cargo molecule is at least one selected from the group consisting of a nucleic acid; peptide; protein; peptide-nucleic acid; oligosaccharide; lipid; glycolipid; lipoprotein; small molecule compound; therapeutic drug; UV-vis, fluorescent or radioactive label; imaging agent; diagnostic agent; prophylactic agent; liposome; and virus.
In various embodiments, the at least one cell is human.
In various embodiments, the at least one cell is in a human subject.
In various embodiments, the contacting comprises administering a pharmaceutical composition comprising an effective amount of the transport construct and at least one pharmaceutically acceptable carrier to the human subject.
In another aspect, the invention provides an isolated transport construct comprising a cargo molecule and at least one cell-penetrating peptide selected from the group consisting of SEQ ID NOs: 165-517, or a salt or solvate thereof.
In various embodiments, the cell-penetrating peptide is SEQ ID NO: 2.
In various embodiments, the transport construct further comprises at least one selected from the group consisting of an activity modulating flanking sequence, a temperature modulating flanking sequence, and a pH modulating flanking sequence.
In various embodiments, the transport construct further comprises a linker connecting the cargo molecule and the cell-penetrating peptide.
In various embodiments, the cargo molecule is at least one selected from the group consisting of a nucleic acid; peptide; protein; peptide nucleic acid; oligosaccharide; lipid; glycolipid; lipoprotein; small molecule compound; therapeutic drug; UV-vis, fluorescent or radioactive label; imaging agent; diagnostic agent; prophylactic agent; liposome; and virus.
In various embodiments, the invention provides a pharmaceutical composition comprising the transport construct and at least one pharmaceutically acceptable excipient.
In another aspect, the invention provides a method of preventing viral infection in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of a polypeptide comprising a cell-penetrating peptide and a retromer binding site.
In various embodiments, the viral infection is papillomavirus, hepatitis C virus, influenza virus or human immunodeficiency virus (HIV).
In various embodiments, the therapeutically effective amount of the polypeptide is formulated for topical administration.
In various embodiments, the polypeptide comprises at least one cell-penetrating peptide selected from the group consisting of SEQ ID NOS: 165-517.
In various embodiments, the polypeptide comprises a retromer binding site comprising the sequence FYL.
The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, certain embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, selected materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, subcutaneous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
As used herein, the term “cell-penetrating peptide” or “CPP” refers to a cell-permeable peptide, which is defined as a peptide capable of permeating and/or crossing a cell membrane. CPPs are sometimes referred to as protein transduction domains (PTD)
An “effective amount” or “therapeutically effective amount” of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered. An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
“L2” as used herein, refers to the minor capsid protein of a papillomavirus, of which the HPV16 type has the amino acid sequence (SEQ ID NO. 518):
The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.
As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it can perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds can also be incorporated into the compositions. The “pharmaceutically acceptable carrier” can further include a pharmaceutically acceptable salt of the compound useful within the invention. Other additional ingredients that can be included in the pharmaceutical compositions used in the practice of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.
As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide (or amide) bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
As used herein, the term “solvate” refers to a complex between a molecule and a solvent molecule, which can exist in solution or in solid phase. In certain embodiments, the solvent comprises at least one selected from the group consisting of water, methanol, ethanol, n-propanol, 2-propanol, DMSO, DMF, ethyl ether, acetone and pyridine.
As used herein, the term “transport construct” refers to a construct that crosses the cell membrane, wherein the construct comprises a cell-penetrating peptide and at least one heterologous cargo molecule, wherein the cargo molecule alone crosses the cell membrane at a lower rate or to a lower degree than the transport construct. In certain embodiments, the cargo molecule is selected from the group consisting of a nucleic acid; peptide; protein; peptide nucleic acid; oligosaccharide; lipid; glycolipid; lipoprotein; small molecule compound; therapeutic drug; UV-vis, fluorescent or radioactive label; imaging agent; diagnostic agent; prophylactic agent; liposome and virus. In other embodiments, the cargo molecule is linked to the transport peptide through a covalent or non-covalent linkage.
As used herein, “treating a disease or disorder” means reducing the frequency or severity with which a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.
As used herein, the term “treatment” or “treating” encompasses prophylaxis and/or therapy. Accordingly the compositions and methods of the present invention are not limited to therapeutic applications and can be used in prophylactic ones. Therefore “treating” or “treatment” of a state, disorder or condition includes: (i) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a subject that can be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (ii) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (iii) relieving the disease, i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
The invention is based in part on the discovery that the human papillomavirus L2 protein C-terminal basic sequence acts as a cell-penetrating peptide that facilitates entry into the cell and escape from the endosome. These sequences are unlikely to be toxic because they naturally evolved to support infection by a non-lytic virus. There are a variety of analogous peptides found in various types of papillomavirus that can be fused to a cargo molecule and used for cell penetration and endosomal escape.
In one aspect, the invention provides an isolated transport construct comprising a cargo molecule and at least one cell-penetrating peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 164, or a salt or solvate thereof. In various embodiments, the cell-penetrating peptide is SEQ ID NO: 2, which is RKRRKR. This is the basic sequence common to several papillomavirus L2 proteins including HPV16 and is evaluated in the examples described elsewhere herein. The sequences for other cell-penetrating peptides are shown in Table 1.
In various embodiments, the cargo molecule is selected from the group consisting of a nucleic acid; peptide; protein; peptide nucleic acid; oligosaccharide; lipid; glycolipid; lipoprotein; small molecule compound; therapeutic drug; UV-vis, fluorescent or radioactive label; imaging agent; diagnostic agent; prophylactic agent; liposome; and virus.
The cargo molecule can be combined with or linked to the cell-penetrating peptide to form the transport construct of the present invention. The cell-penetrating peptide and the cargo molecule are combined or linked in such a manner that they remain combined or linked under the conditions in which the transport construct is used (e.g., under conditions in which the transport construct is administered to an individual).
In certain embodiments, the cargo molecule is covalently linked to the cell-penetrating peptide through a linker or a chemical bond. In other embodiments, the linker comprises a disulfide bond, or the chemical bond between the cargo molecule and the transport peptide comprises a disulfide bond. In yet other embodiments, the cargo molecule comprises a peptide. In yet other embodiments, the transport peptide is covalently linked through an amide bond to the N-terminus of the peptide molecule of the cargo molecule. In yet other embodiments, the transport peptide is covalently linked through an amide bond to the C-terminus of the peptide of the cargo molecule. In yet other embodiments, the transport peptide is covalently linked through an amide bond to both the N-terminus and the C-terminus of the peptide of the cargo molecule.
Alternatively, the transport peptide and the cargo molecule are combined through a noncovalent linkage, such as electrostatic and/or hydrophobic interaction.
In various embodiments, the transport construct further comprises a linker connecting the cargo molecule and the cell-penetrating peptide. In various embodiments, the linker is a disulfide, a polyethylene glycol chain (PEG), a short polypeptide chain, or an amide, or other linkers that do not impair activity. In various embodiments, the cargo molecule can inhibit infection by papillomavirus. As shown in
In various embodiments, the transport construct can include one or more flanking sequences that modulate the cell-penetrating activity or properties of the transport construct. In various embodiments, the flanking sequences can be an activity modulating flanking sequence, a flanking sequence that modulates temperature dependence, and/or a flanking sequence that modulates pH dependence, or a flanking sequence that modulates other aspects of cell penetration, such as lipid composition of the membrane. The term “flanking sequence” as used herein refers to a peptide fused to the transport construct that modulates the cell-penetrating activity of the transport construct. In various embodiments, the flanking sequence can modulate the cell-penetrating activity of the transport construct generally. As shown in
In various embodiments, the flanking sequence can be a certain portion of L2 from a form of papillomavirus selected to modulate the activity of the transport construct based on the characteristics of the papillomavirus. As shown in
In various embodiments, the invention provides a pharmaceutical composition comprising the transport construct as described herein and at least one pharmaceutically acceptable excipient.
In another aspect, the invention provides a method of delivering a cargo molecule to the cytoplasm of at least one cell by contacting the cell with a transport construct comprising the cargo molecule and at least one cell-penetrating peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 164, or a salt or solvate thereof.
In another aspect, the invention provides a method of promoting endosomal escape for a cargo molecule by contacting at least one cell with a transport construct comprising the cargo molecule and at least one cell-penetrating peptide selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 164. In various embodiments, the cell-penetrating peptide is SEQ ID NO: 2, or a salt or solvate thereof.
The cargo molecule can be any molecule intended for delivery to the cytoplasm and that can be covalently linked the cell-penetrating peptides of the invention. In various embodiments, the cargo molecule is at least one selected from the group consisting of a nucleic acid; peptide; protein; oligosaccharide; lipid; glycolipid; lipoprotein; small molecule compound; therapeutic drug; UV-vis, fluorescent or radioactive label; imaging agent; diagnostic agent; prophylactic agent; liposome; and virus.
The methods of the invention can be applied to deliver a cargo molecule to cells in vitro or to the cells of a subject. In various embodiments, the contacting step comprises administering a pharmaceutical composition comprising the transport construct and at least one pharmaceutically acceptable carrier to a subject. In various embodiments, the cell or cells are human. In various embodiments, the subject is human. Various suitable pharmaceutically acceptable carriers are described under administration/dosage/formulations.
In table 1, the left two columns list virus type and associated cell penetrating peptide. The right two columns compile the basic sequences found in the various virus-associated cell-penetrating peptides and their frequency.
Without wishing to be limited by theory, it has now been found that delivering a peptide containing a retromer binding site to a cell in advance of or shortly after exposure to a virus prevents viral infection by sequestering retromer and interfering with the role this complex plays in the viral infection of the cell. Accordingly, in another aspect, the invention provides a method of preventing viral infection in a subject in need thereof, the method comprising providing to the subject a therapeutically effective amount of a polypeptide comprising a cell-penetrating peptide and a retromer binding site. In various embodiments, the viral infection is papillomavirus, hepatitis C virus, influenza virus or human immunodeficiency virus (HIV). The polypeptide is provided to the subject by a method appropriate for the viral infection to be prevented. In various embodiments, the polypeptide is formulated for topical administration.
The cell-penetrating peptide may be any cell-penetrating peptide known in the art including but not limited to the cell-penetrating peptides taught herein. In various embodiments, the polypeptide comprises at least one cell-penetrating peptide selected from the group consisting of SEQ ID NOS: 1-164. In various embodiments, the polypeptide comprises at least one cell-penetrating peptide selected from the group consisting of SEQ ID NOS: 165-517. In various embodiments, the polypeptide comprises a retromer binding site comprising the sequence FYL.
The regimen of administration can affect what constitutes an effective amount for the treatment of various diseases, depending on the nature of the cargo molecule. The therapeutic formulations can be administered to the subject either prior to or after the onset of diseases contemplated herein. Further, several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the therapeutic formulations can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, can be carried out using known procedures, at dosages and for periods of time effective to treat the disease in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect can vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat the disease in the patient. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.
A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of an inflammatory disease in a patient.
The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
In certain embodiments, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.
Compounds of the invention for administration can be in the range of from about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about 40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg to about 7,500 mg, about 200 μg to about 7,000 mg, about 350 μg to about 6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.
In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.
In certain embodiments, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of the disease in a patient.
Formulations can be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They can also be combined where desired with other active agents, e.g., other analgesic agents.
Routes of administration of any of the compositions of the invention include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention can be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.
Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.
For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use can be prepared according to any method known in the art and such compositions can contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets can be uncoated or they can be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.
The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of certain diseases or disorders. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition can be obtained in which the active ingredient is entrapped, ensuring its delayed release.
For parenteral administration, the compounds of the invention can be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents can be used.
Additional dosage forms of this invention include dosage forms as described in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this invention also include dosage forms as described in PCT Applications Nos. WO 03/35041; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.
In certain embodiments, the formulations of the present invention can be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time can be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
For sustained release, the compounds can be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention can be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
In one embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
The therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of an inflammatory disease in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.
A suitable dose of a compound of the present invention can be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose can be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage can be the same or different. For example, a dose of 1 mg per day can be administered as two 0.5 mg doses, with about a 12-hour interval between doses.
It is understood that the amount of compound dosed per day can be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose can be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.
The compounds for use in the method of the invention can be formulated in unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.
Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.
It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present application.
The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.
The materials and methods employed in practicing the following examples 1-9 are here described:
HeLa S3 cervical carcinoma cells were obtained from the American Type Culture Collection (ATCC). HeLa-Sen2, a cloned strain of HeLa cells, infect efficiently with SV40 and HPV16 pseudovirus and were used for immunofluorescence experiments. HaCaT adult human keratinocyte cells were purchased from AddexBio. The HPV16 L2 C-terminal mutants were constructed in pHPV16sheLL by using the Phusion site-directed mutagenesis protocol (New England Biolabs, Ipswich, MA). This method was also used to replace the basic segment of the L2 protein with known CPPs: cationic (SEQ ID NO: 519 RKKRRRQRRR), amphipathic (SEQ ID NO: 520 PLSSIFSRIDG), and hydrophobic (SEQ ID NO: 521 AAVLLPVLLAAP), from HIV Tat, hepatitis B virus large surface antigen, and the K-fibroblast growth factor signal peptide, respectively. The L1 and L2 genes in each mutant were sequenced to verify mutations.
HPV PsVs were produced by co-transfecting 293TT cells with pCAG-HcRed (cat. #11152; Addgene, Cambridge, MA) and p16sheLL or p16L1-GFP with or without mutations or a FLAG tag at the C-terminus of L2. The packaged PsVs were purified by density gradient centrifugation in Opti Prep (cat. ##1114524; Axis-shield, Dundee, Scotland). Encapsidated HcRed genomes were quantified by qPCR as described. Briefly, purified PsVs were treated with DNase I (cat. #79254; Qiagen, Germantown, MD) to remove unencapsidated DNA associated with capsids. Reporter DNA genome was isolated using a DNA purification kit (Qiagen, #69504), and the copy number of encapsidated reporter plasmids was determined by qPCR using primers for the HcRed gene in comparison to a standard curve.
Five μl of pseudovirus solution was placed on freshly glow-discharged copper grids (formvar/carbon-coated, 200 mesh, Electron Microscopy Services, Hatfield, PA). After two min, the grids were rinsed twice with droplets of deionized water, stained by 2% aqueous uranyl acetate for two min, and then the excess staining solution was blotted off. The grids were allowed to air dry for 15 min. The samples were examined in a FEI Tecnai F20 transmission electron microscope at 200 kV. Images were acquired using a FEI Eagle CCD camera (4K×4K). Virus stocks containing ˜1×107 PsV genomes were analyzed by SDS-PAGE, followed by staining with Coomassie brilliant blue.
5×104 HeLa S3 or HaCaT cells in 12-well plates were incubated with PsVs at MOI of approximately one. The number of packaged reporter plasmids required to achieve this MOI as assessed by flow cytometry for reporter gene expression from wild-type PsV was quantified by qPCR, and an equivalent number of genomes in mutant PsV were used to infect cells. Approximately five-fold more virus was used to attain this MOI in HaCaT cells. Cells were analyzed by flow cytometry on a Stratedigm-12 flow cytometer to determine the fraction of cells displaying HcRed fluorescence 48 h.p.i. To measure dependence on retromer, 1×105 HeLa S3 cells in a 12-well plate were transfected 48 h.p.i. prior to infection with 10 pmol siRNAs targeting Vps29 (Dharmacon, Lafayette, CO; #D-009764-03) or Vps35 (Dharmacon, #D-010894-01) by using RNAiMAX (Thermo Fisher, Waltham, MA, #13778100) according to manufacturer's protocol. Cells transfected with a 10 pmol RISC-free siRNA were used as a control. Forty-eight h.p.i., infectivity was measured by flow cytometry for reporter gene expression. To measure dependence on γ-secretase, HeLa S3 cells were pretreated for one h with 250 nM γ-secretase inhibitor, compound XXI, which was retained in the medium for the duration of the experiment. Cells were infected at MOI of one. Forty-eight h.p.i., infectivity was measured by flow cytometry for HcRed expression. To measure the effect of aphidicolin, HeLa S3 cells were pretreated for 24 h with 6 μM aphidicolin, which was retained in the medium for the duration of the experiment. Cells were infected at MOI of one. Forty-eight h.p.i., infectivity was measured by flow cytometry for HcRed expression.
To measure cell binding, 5×104 HeLa-Sen2 cells grown on glass coverslips were infected with wild-type PsV at MOI of 20 or mutant PsV containing the same number of encapsidated reporter plasmids. Cells were incubated with PsVs at 4° C. for two h and then washed three times with phosphate-buffered saline (PBS). Cells were fixed for 15 min at room temperature with 4% Formalde-Fresh, washed with PBS and reacted with anti-L1 rabbit polyclonal serum (obtained from J. Schiller, NIH), followed by fluorescently-tagged donkey anti-rabbit secondary antibody. Images were acquired with a Leica SP5 inverted fluorescence microscope and processed with ImageJ.
For the internalization experiments, HeLa cells were incubated with PsV at MOI of 50 at 4° C. for two h, followed by extensive washing in PBS to remove loosely bound PsV. Cells were then shifted to 37° C. to initiate internalization. After six hours, samples were fixed, permeabilized and reacted with anti-L1 antibody (BD Biosciences, San Jose, CA, #554171), followed by donkey anti-rabbit secondary antibody. Cells were analyzed by fluorescence confocal microscopy. In the experiment shown in
For the proximity ligation assay, HeLa-Sen2 cells were infected with wild-type PsV at MOI of 100-to-200 or mutant PsV containing the same number of encapsidated reporter plasmids. Infected cells were fixed, permeabilized at various times post-infection, and incubated with pairs of antibodies, one recognizing L1 (#554171) and the other recognizing a cellular marker or a retromer subunit. PLA was performed with Duolink reagents from Olink Biosciences (Uppsala, Sweden) according to the manufacturer's directions. Briefly, cells were incubated in a humidified chamber with a pair of suitable PLA antibody probes diluted 1:5 and processed for ligation and amplification with fluorescent substrate at 37° C. Images were acquired as described above. Approximately 100 nuclei in each sample were imaged. The images were processed by ImageJ and quantitatively analyzed by BlobFinder software to measure total fluorescence intensity in each sample. The average fluorescence intensity per cell in each sample was normalized to the control sample as indicated in each experiment. Similar results were obtained in three independent experiments for each antibody pair.
In all fluorescence imaging experiments, cells were also stained with DAPI or Hoechst 33342 to visualize nuclei (blue), and a single confocal Z-plane is shown in each panel.
To measure cell surface binding by wild-type and mutant PsV, 7.5×105 HeLa cells in six-well plates were incubated with wild-type PsVs at MOI of 20 or mutant PsVs containing the same number of reporter plasmids at 4° C. for two h and then washed three times with PBS. Cells were harvested by treatment of 0.5 mM EDTA on ice for 15 min. Samples were fixed in ice-cold methanol and stained with mouse anti-L1 polyclonal IgG (BD Biosciences, #554171) and incubated with corresponding Alexa Fluor secondary antibodies. Fluorescence intensity was assayed on a Stratedigm-13 flow cytometer.
To measure internalization, 7.5×105 HeLa cells in six-well plates were incubated at 4° C. for two h with wild-type PsV at MOI of 20 or mutant PsV containing the same number of reporter plasmids and then washed three times with PBS, followed by additional incubation at 37° C. for six h to initiate internalization. Cells were then harvested by trypsinization to remove PsV on the cell surface. Samples were fixed in ice-cold methanol and stained with anti-L1 antibody #554171 and incubated with Alexa Fluor-labeled secondary antibody. Fluorescence intensity was assayed on a Stratedigm-13 flow cytometer.
5×104 293T cells grown on eight-chambered glass coverslips were incubated with 30 μM fluorescently-labeled peptides for three hours at 37° C. or mock-infected. 5×104 HaCaT and HeLa S3 cells grown on eight-chambered glass coverslips were incubated with 2.5 μM wild-type or mutant His-tagged GFP-L2 fusion proteins (see next section) for three h at 37° C. After treatment, cells were washed with PBS three times, stained with Hoechst 33342, and examined by confocal microscopy. To observe internalized fusion proteins, cells were treated for 10-15 min with 0.04% trypan blue, washed with PBS, and examined by confocal microscopy.
For fusion protein pull-down experiments, individual human Vps26, Vps29, and GST-tagged Vps35 subunits were expressed individually in E. coli, and the assembled trimeric retromer complex was immobilized on GSH resin (GE Health Care Life Sciences, Pittsburgh, PA, #17075601). His6-GFP-L2 fusion proteins containing a C-terminal segment from wild-type or mutant HPV16 L2 (amino acids 435-461) were expressed in bacteria and purified using the His GraviTrap column (GE Healthcare, #11-0033-99). Purified proteins were exchanged into PBS buffer by dialysis and quantified by bicinchoninic acid protein assay. Ten g fusion protein was incubated with assembled 15 μg retromer trimer immobilized on GSH resin for two h at 4° C. in 20 mM HEPES pH 8.0, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 0.1% Triton X-100. Beads were centrifuged and washed twice in HEPES buffer, suspended in SDS loading buffer, boiled, and subjected to SDS-PAGE and anti-GFP immunoblotting.
For peptide pull-down experiments, N-terminal biotinylated L2 peptides (sequences shown in
Plasmids. The full-length GFP gene in pLenti CMV GFP Puro (658-5) (Addgene, #17448) contained on a BamHI and SalI fragment was replaced with the DNA segment encoding GFP1-10 amplified from pCMV-mGFP1-10 to generate pLenti CMV.GFP1-10. To construct pLentiCMV.GFP1-10NES, oligonucleotides encoding the nuclear export signal (NES) sequence LPPLERLTLD (SEQ ID NO: 522) were inserted in-frame at the C-terminus of GFP1-10 in pLentiCMV.GFP1-10.
Plasmid pCD8-CIMPR expresses a CD8-CIMPR fusion protein containing the extracellular domain of CD8 fused to the transmembrane and mutant cytoplasmic domain of the cation-independent mannose phosphate receptor (CIMPR), which contains an endocytosis motif and three alanines replacing the WLM retromer binding site. The In-Fusion HD cloning Kit (TaKaRa Bio USA, Inc., Kusatsu, Shiga, Japan), #121416) was used to insert seven tandem copies of GFP11 (SEQ ID NO: 523 RDHMVLHEYVNAAGIT) in-frame into pCD8-CIMPR either immediately downstream of the CD8 signal peptide sequence or at the C-terminus of the fusion protein to generate pGFP11-CD8-CIMPR and pCD8-CIMPR-GFP11, respectively.
Each GFP 11 repeat was separated by short GGSGG (SEQ ID NO: 524)linker sequences. The sequences of the relevant portions of the encoded CD8-CIMPR fusion proteins are as follows, with GFP11 and linker sequences in bold.
MVLHEYVNAAGITGGKSQFRV...
LHEYVNAAGITGGK.
Plasmids CNX-S1-10(N) and CNX-S1-10(C) were obtained from Bernard Moss (NIH) and designated here GFP1-10-CNX and CNX-GFP1-10, respectively.
To construct pHPV16sheLL-GFP11-CPP for producing PsVs containing GFP11 inserted into the HPV16 L2 protein immediately upstream of the basic segment, DNA encoding the tandem GFP11 repeats was amplified from pGFP11-CD8-CIMPR by using the In-Fusion HD cloning Kit and cloned in-frame into L2 in p16sheLL. pHPV16sheLL-CPP-GFP11 with the GFP 11 repeats at the extreme C-terminus of L2 was constructed by first inserting Nsil and AvrlI restriction sites immediately upstream of the L2 stop codon in p16sheLL. The DNA segment encoding the GFP11 repeats was then inserted into this plasmid between these sites. Site-directed mutagenesis of pHPV16sheLL-GFP11-CPP and pHPV16sheLL-CPP-GFP11 was used to construct pHPV16sheLL-GFP11-3R and pHPV16sheLL-3R-GFP11, respectively, both of which contain three arginines in place of the wild-type CPP. The sequences of the relevant portions of the encoded L2-GFP 11 fusion proteins with wild-type CPP are as follows, with GFP 11 and linker sequences in bold and L2 CPP underlined.
GITGGKF
RKRRKRLPYFFSDVSLAA.
TGGSGG)6RDHMVLHEYVNAAGITGGKFPRL.
Generation of stable cell lines. To generate stable cell lines expressing cytoplasmic GFP1-10NES, lentiviruses were produced by co-transfection of 293T cells in 100 mm dishes with 6 μg of pLentiCMV.GFP1-10NES, 4.5 μg of lentiviral packaging plasmid psPAX2 and 1.5 μg envelope plasmid pMD2.G. Forty-eight h later, the lentiviral supernatant was harvested, filtered and stored at −80° C. for later use. Cells stably expressing GFP1-10NES were constructed by infecting HaCaT cells with lentivirus for 48 h in medium containing 1 μg/mL puromycin. Single cells were then plated in a 96-well plate, and monoclonal cell lines were isolated and tested by infection of HPV16-CPP-GFP11 PsV at ˜2000 MOI and monitoring fluorescence.
Immunofluorescence microscopy of GFP1-10 expression. 5×104 clonal HaCaT/GFP1-10NES stable cells grown on glass coverslips were fixed for 15 min at room temperature with 4% Formalde-Fresh, washed with PBS and incubated with anti-GFP mouse antibody (Santa Cruz, Dallas, TX, #SC-9996) and Alexa Fluor 488 donkey anti-mouse IgG (H&L) secondary antibody (Fisher, #A-21202). Images were acquired with a Leica SP5 inverted fluorescence microscope to identify cell lines expressing cytoplasmic GFP1-10NES.
Validation of the split GFP assay. 5×104 clonal HaCaT/GFP1-10NES cells in eight-chambered glass slides were transfected with 0.25 μg of pGFP11-CD8-CIMPR or pCD8-CIMPR-GFP11. Twenty-four hours later, live cells were examined by fluorescence microscopy. To demonstrate the functionality of GFP11 in GFP11-CD8-CIMPR, 5×104 293T cells in eight-chambered glass slides were co-transfected with a plasmid expressing GFP11-CD8-CIMPR and a plasmid expressing a calnexin transmembrane fusion protein containing luminal GPF1-10 or cytoplasmic GFP1-10 (GFP1-10-CNX and CNX-GFP1-10, respectively) (
Use of split GFP assay to demonstrate cytoplasmic protrusion of L2. 3×104 clonal HaCaT/GFP1-10NES cells seeded in eight-chambered glass slides were incubated for 1.5 or 3 h at MOI of ˜2000 with HPV16 PsV containing wild-type L2 or L2 containing inserted GFP11 and a wild-type or 3R mutant CPP. Live cells were analyzed by a Leica SP5 inverted fluorescence microscope and processed with ImageJ. In the experiment shown in
Fusion proteins with various segments of the C-terminus of HPV16 or HPV5 L2 were added to HaCaT cells. The sequences used are as follows:
In some cases, various concentrations of fusion proteins were added to assess efficacy. In other cases, the pH of the culture medium was physiological pH 7 or adjusted to acidic pH 4 prior to peptide addition, or the cells were incubated with peptide at physiological temperature of 37° C. or at 30° C. Activity was assessed by confocal microscopy for intracellular fluorescence. In some experiments, cell surface fluorescence was extinguished by treatment with the membrane-impermeable fluorescence quencher, trypan blue.
HeLa cells were left untreated or treated at 37° C. with 4 μg L2-C, a peptide containing the HPV16 L2 retromer binding site and CPP. The sequence of L2-C is as follows: B-SPQYTIIADAGDFYLHPSYYMLRKRRKR-Am, (SEQ ID NO: 533), where B represents N-terminal biotinylation and Am represents C-terminal amidation. Two hours later, the cells were infected for two hours at 37° C. with wild-type HPV16 pseudovirus. Cells were washed and medium was replenished with medium containing the peptide for 24 hours. Infectivity was tested 48 hours later by using flow cytometry to measure fluorescence of HcRed expressed from a reporter plasmid encapsidated in the pseudovirus particle, and normalized to infectivity of cells infected in the absence of peptide. Similar results were obtained in two independent experiments.
Direct binding of retromer to a carboxy-terminal segment of the HPV16 L2 protein is required for transport of the incoming virus from endosomes to the Golgi. To test whether the basic amino acids in the C-terminus of the L2 protein function as a CPP to transfer a segment of the L2 protein into the cytoplasm to allow a direct interaction with retromer, it was first tested whether mutations in this segment of L2 inhibited infectivity. For these experiments, pseudoviruses (PsVs) comprised of an HcRed reporter plasmid, wild-type HPV16 L1, and wild-type or mutant HPV16 L2 were used. PsV assembly was confirmed by electron microscopy, which showed no obvious morphologic differences between wild-type and mutant PsVs (
To determine if the L2 basic sequence is important for HPV16 infection, wild-type RKRRKR (SEQ ID NO: 2) was replaced with six alanines (6A mutant) (
RKRRKR (SEQ ID NO: 2) was then replaced with six consecutive lysines or with various numbers of arginines (
It was next tested if the HPV16 PsV containing L2-Tat utilized a similar entry pathway as wild-type HPV16 PsV. To determine if retromer was required for infection mediated by L2-Tat, cells transfected with siRNA that knocked down the Vps29 retromer subunit were infected.
Retromer knockdown dramatically inhibited infection by either wild-type or the L2-Tat PsV, indicating that the L2-Tat chimera requires retromer (
Experiments were conducted to determine whether the basic sequence of L2 has intrinsic cell penetrating activity. First, Alexa Fluor 488 was conjugated onto the N-terminus of a 28-residue L2 peptide that terminates with the wild-type basic sequence or the corresponding L2 peptide with the 6A or the 3R mutation (
To confirm CPP activity of this L2 segment, GFP fusion proteins were expressed and purified from bacteria consisting of GFP fused in-frame to a 28-residue segment from the C-terminus of L2 terminating with either the wild-type basic segment or a mutant segment (
To determine the role of the L2 CPP in HPV infection, cell binding experiments were conducted. HeLa cells were incubated with either wild-type or mutant HPV16 PsV for two hours at 4° C., followed by washing to remove unbound viruses. Non-permeabilized cells were stained with an L1 antibody, and immunofluorescence was performed to detect viruses stably bound to cells. As shown in
The role of the L2 CPP in virus internalization was examined next. After incubation of cells with HPV16 PsVs at 4° C., cells were shifted to 37° C. for six hours to allow internalization. Internalization was assessed by immunofluorescence and by flow cytometry. The 3R L2 mutant internalized into cells as well as wild-type, while the 6A mutant showed much less internalized L1, as expected because of its cell surface binding defect (
To examine the post-internalization defect of the 3R mutant, proximity ligation assay (PLA) was used to determine the localization of incoming wild-type and 3R mutant HPV16 PsV. PLA is an immune-based assay used to test if two proteins of interest are within 40 nm. PLA was performed with an anti-L1 antibody and an antibody that recognizes either EEA1, a marker of the early endosome, or the trans-Golgi network (TGN) marker, TGN46. The L2 double mutant (DM), which lacks retromer binding sites, was used as a control. As shown in
It was next tested whether the 3R mutation impaired association between the capsid and retromer in infected cells. HeLa cells were infected with either wild-type or mutant HPV16 PsV, and PLA was performed with an anti-L1 antibody and an antibody recognizing Vps35, a subunit of retromer. As shown in
In certain embodiments, the 3R mutation can inhibit retromer association directly, by impinging on the retromer binding sites, or indirectly, by preventing the exposure of the binding sites in the cytoplasm. To determine if mutations in the L2 CPP directly inhibited binding to retromer, pull-down experiments were performed. GST-tagged retromer subunits were expressed in bacteria, assembled into the trimeric retromer complex, and bound to glutathione beads. The purified GFP-L2 fusion proteins containing a wild-type L2 segment, the 3R mutation, or the DM mutations in the retromer binding sites were incubated with the retromer beads at 4° C., pelleted, and subjected to western blotting with an anti-GFP antibody to detect the L2 fusion protein in the pellet. As expected, the wild-type protein bound retromer well and the DM fusion protein bound poorly (
To directly demonstrate membrane passage of the L2 C-terminus during virus entry, a split GFP imaging method was adapted. A protein consisting of GFP beta strands 1 to 10 (GFP1-10) does not fluoresce, nor does the 16-residue eleventh beta strand of GFP (GFP11). However, when GFP11 is in the same cellular compartment as GFP1-10, holo-GFP is reconstituted, generating a fluorescent signal. This approach has been used to demonstrate cytoplasmic delivery of soluble fusion proteins linked to CPPs.
To use this assay to assess L2 exposure, a clonal HaCaT cell line expressing GFP1-10 linked at its C-terminus to a nuclear export signal (NES) was constructed (
To assess cytoplasmic exposure of L2 during infection, seven tandem copies of GFP11 were inserted at two different positions in the C-terminus of the L2 protein: between the CPP and the retromer binding sites (L2-GFP11-CPP) or at the extreme C-terminus of L2 (L2-CPP-GFP11) (
To test if the L2 CPP was required for membrane protrusion, the wild-type CPP in L2-GFP11-CPP and L2-CPP-GFP11 was replaced with three arginines (
To assess the effect of sequences flanking the core basic CPP, fusion proteins were constructed and purified with varying portions of the C-terminal segment of HPV16 and HPV5 L2. HPV16 typically infects genital and oral epithelia, at body core temperature of 37° C., whereas HPV5 is a virus that infects skin, which is several degrees cooler. The ability of these L2 segments to allow the fusion proteins to bind to cells and be delivered intracellularly was tested under a variety of conditions. In aggregate, these experiments show 1) that the 21 amino acids upstream of the HPV16 L2 CPP stimulates the ability of fusion proteins to enter cells (
A major long-term goal is to use CPPs to deliver bioactive peptides into cells as therapeutics. As an example of using the HPV L2 CPP to deliver a novel peptide-based inhibitor that blocks HPV infection. Intracellular delivery of peptides containing the retromer binding site (RBS) of HPV16 compete for retromer binding with the RBS on the L2 protein of the incoming virus particle protruding through the endosome membrane, and thus cause HPV to accumulate in the endosome. Cells were treated with peptide L2-C, comprised of a C-terminal segment of HPV16 L2 including the RBS and adjacent CPP. The CPP delivers the peptide across the plasma membrane into the cytoplasm where the RBS on the peptide will bind retromer and compete with the intact L2 protein on the incoming virion, thereby inhibiting infection.
In order to traffic to the nucleus, papillomaviruses rely on cellular retrograde transport, but it was not clear how the incoming virion in the endosomal lumen enters the retrograde pathway. Here, these data show that a short sequence of basic amino acids near the C-terminus of the L2 protein acts as a CPP to transfer a segment of the L2 protein into the cytoplasm where adjacent sequences can bind retromer for transport to the TGN. First, it was shown that the basic region of L2 is required for efficient infection of epithelial cells and can be replaced with the cationic CPP from HIV-1 Tat. Like wild-type HPV16 PsV, PsV containing the Tat CPP required retromer and γ-secretase for infection. Five or more consecutive arginine residues restored full infectivity, whereas fewer arginines and six lysines were less effective, consistent with the known cell-penetrating activities of these sequences. Peptide and protein transduction assays were used to demonstrate that the basic segment of L2 did in fact display CPP activity. Importantly, a truncated sequence of three arginines was defective for CPP activity and failed to support infection. The preferential uptake of the 3R peptide compared to the 3R fusion protein may reflect the much higher concentration of molecules used in the peptide experiments. Taken together, these experiments demonstrated that L2 CPP activity was required for HPV infection.
The presence of a C-terminal basic region in all papillomavirus L2 proteins implies that the essential role of the L2 CPP has been maintained since the papillomaviruses first emerged more than 250 million years ago. The amino acid sequence of the basic segment is variable, consistent with the relatively relaxed sequence requirements for CPPs. The 353 sequenced L2 proteins in the papillomavirus PaVe sequence database (pave dot niaid dot nih dot gov/#home) contain 164 different C-terminal basic sequences, including a 10-residue poly-arginine stretch in three canine viruses (Table 1). RKRRKR (SEQ ID NO: 2) present in HPV16 L2 is one of the most common, being found in 16 diverse human and animal papillomaviruses, and many more CPPs are likely to exist in the papillomavirus virome because most of these different basic sequences have been identified in only a single virus type (Table 1). In addition, sequences flanking the core basic amino acids may influence membrane penetration activity. The multitude of papillomavirus types thus represents the results of a mutational analysis carried out over evolutionary time, revealing hundreds of different, presumably non-toxic sequences that can have cargo-carrying activity.
HPV16 PsV containing a mutant CPP consisting of three arginines was internalized, showing the L2 CPP was not required for endocytosis, but the mutant was defective for retromer engagement, exit of the virus from the endosome, and trafficking to the TGN. The same phenotype is caused by retromer binding site mutations. However, the CPP mutation did not directly impair the ability of L2 to bind retromer, implying that during infection the retromer binding sites were not in the same cellular compartment as retromer.
To directly assay the cytoplasmic exposure of L2, a split GFP assay was developed, in which fluorescence is reconstituted when a segment of GFP at the C-terminus of L2 encounters GFP1-10 in the cytoplasm. Cytoplasmic exposure of L2 was detectable early during infection and was impaired by replacing the CPP with three arginines. These findings show that the L2 CPP mediates passage of the C-terminus of the L2 protein into the cytoplasm so that it can engage retromer and enter the retrograde trafficking pathway. In some cells, reconstituted fluorescence is fairly uniform throughout the cytoplasm (
The rapid generation of reconstituted GFP fluorescent signal is consistent with the fact that the L2 protein binds to the cytoplasmic protein SNX17 as early as two h.p.i. In addition, by one and a half to three h.p.i., intracellular L1 is detectable by immunofluorescence with the 33L1-7 conformation-specific antibody (
The physiological role of most CPPs is not known. Naturally-occurring CPPs are usually studied as small peptide fragments removed from their protein of origin, and in many cases cell-penetrating activity may be the fortuitous consequence of basic sequences with no natural role in membrane penetration. These results show that CPP-driven membrane penetration by L2 plays an important role in HPV infection and that the L2 CPP of endocytosed virus protrudes through the endosomal membrane into the cytoplasm. The bulk of L2 then passes through the membrane, possibly assisted by endosomal acidification, until it is arrested by its N-terminal transmembrane domain in a type 1 transmembrane orientation with its N-terminus in the endosomal lumen and most of the protein exposed in the cytoplasm. The exposed C-terminus then binds to essential entry factors, including retromer, which sorts the virus into retrograde transport vesicles that later fuse with more distal retrograde compartments. The effect of the L2 CPP on the membrane is relatively subtle and localized, in contrast to the more drastic membrane disruption events caused by other non-enveloped viruses, which are deposited into the cytoplasm. This allows the residual HPV virion, including viral DNA, to be retained in transport vesicles and presumably contributes to the relatively low immunogenicity of these viruses during cell entry by sequestering them from cytoplasmic immune sensors. The L2 protein may protrude into the cytoplasm in a sequential fashion, with the C-terminus being exposed prior to the middle of the protein. Thus, cellular proteins may bind L2 sequentially, first retromer to the C-terminus of L2 and later proteins such as SNX17 to the middle portion of L2. Such ordered binding may be important for the assembly of the protein complexes necessary for proper trafficking. It also is important to note that each capsid contains up to 72 L2 proteins. The presence of multiple L2 molecules in each virion ensures a high local concentration of CPPs upon infection even at low MOI, which may be important for membrane penetration activity. However, it is possible that not all L2 molecules insert into membranes or bind retromer or other cytoplasmic factors.
The experiments reported here show that the L2 protein is an inducible transmembrane protein. In the intact capsid, which lacks membranes, L2 cannot have any transmembrane character. However, once the L2 CPP inserts into the membrane and protrudes into the cytoplasm, the L2 protein adopts a transmembrane existence. Thus, CPPs can not only transport molecules into cells or between compartments, but can also transform a soluble protein into a transmembrane one. Cellular and other viral proteins may also transition from a soluble to a transmembrane state, with important consequences for their biological activities. These results suggest that the primary role of CPPs in biology may not be to transfer proteins into cells in a paracrine fashion but rather to act intracellularly to mediate the transfer of proteins or protein segments between cellular compartments and to convert soluble proteins into transmembrane proteins.
The Materials and Methods employed in Example 10 are here described.
HeLa-S3 cells were obtained from the American Type Culture Collection (ATCC). HaCaT cells purchased from AddexBio Technologies are spontaneously transformed keratinocytes from histologically normal skin. 293TT cells, generated by introducing SV40 Large T antigen cDNA into 293T cells to increase SV40 Large T antigen expression. All cells were cultured in DMEM with HEPES and L-glutamine, supplemented with 10% fetal bovine serum, and 100 units/mL penicillin-streptomycin at 37° C. in 5% CO2.
The plasmids designated p16sheLL, p18sheLL, and p5sheLL, expressing both L1 and L2 for HPV16, HPV18, and HPV5 pseudovirus production, respectively. pCAG-HcRed reporter plasmid was purchased from Addgene.
HPV PsV was produced by co-transfecting 293TT cells with pCAG-HcRed and a psheLL expression plasmid with or without mutations at the C terminus of L2. The packaged PsV was purified by density gradient centrifugation in Opti Prep. Quality of PsV preparations was confirmed by SDS-PAGE, followed by Coomassie brilliant blue staining for L1 and L2. SV40 was prepared in CV1 cells as described.
The infectious multiplicity-of-infection (MOI) of HPV PsV was assessed by flow cytometry for reporter gene expression after infection of HeLa-S3 cells with wild-type HPV16 PsV. The number of packaged reporter plasmids required to achieve the MOI for wild-type PsV was quantified by qPCR, and an equivalent number of genomes in mutant PsV was used to infect cells. For quantifying encapsidated HcRed genomes, purified PsV was treated with DNase I to remove free DNA associated with capsids. Reporter DNA genome was isolated using a DNA purification kit, and the copy number of encapsidated reporter plasmid was determined by qPCR using primers for the HcRed gene in comparison to a standard curve.
Peptides were purchased from ABclonal Technology at >95% purity. Peptide (sequences shown in
To assess the effect of peptides on viral infection, 5×104 HeLa-S3 in 24-well plates were pretreated with peptides for one hour, followed by infection with wild-type HPV PsV at 37° C. at MOI of ˜1 or with mutant PsV containing an equivalent number of encapsidated reporter plasmids. Peptides were left in the medium for the duration of the experiment unless otherwise indicated. As a control, cells were incubated with the solution used to dissolve peptide. Cells were assessed by flow cytometry on a Stratedigm-13 flow cytometer to determine reporter protein expression at 48 h.p.i. unless otherwise indicated. In some experiments, peptides were added at various times after infection and were left in the medium for the duration of the experiment.
To measure the inhibition of infection by authentic HPV16, HeLa cells were infected with 5 μL raft-derived HPV16 (obtained from Craig Meyers, Hershey Medical Center) or with HPV16 PsV in the presence and absence of 14 μM P16/16, and total RNA was isolated 48 h.p.i. by using the RNeasy kit following the manufacturer's instructions. RNA was reverse-transcribed into cDNA by iScript cDNA Synthesis kit. The cDNA was quantified in triplicate by using SYBR Green Supermix real-time PCR detection system and primers for HPV16 E7 or HcRed. Actin mRNA were used for normalization.
For studies of SV40 infection, 7.5×105 HeLa-S3 in 6-well plates were incubated with peptides prior to infection. Cells were infected with a crude preparation of SV40 at MOI of ˜1 and fixed with 4% paraformaldehyde at 48 h.p.i. Samples were stained with FITC-conjugated anti-large T antigen antibody (Pab101) (Santa Cruz, pSC147 FITC), followed by flow cytometry to determine the mean fluorescent intensity of T antigen staining.
For the internalization experiments, 5×104 HeLa-S3 cells were grown on glass coverslips for 16 hours. After one-hour incubation with or without peptides, PsV at MOI of 50 were added and incubated at 4° C. for 2 hours, washed with PBS three times to remove loosely bound PsV, and shifted to 37° C. to initiate infection. As a control, cells were incubated with the solution used to dissolve peptides alone. At the indicated times post-infection, samples were fixed, permeabilized and stained with anti-L1 and AlexaFluor 488 conjugated secondary antibody. Cells were analyzed by a Leica SP5 confocal microscope.
For the proximity ligation assay, after an hour incubation with peptides, HeLa-S3 cells were infected with wild-type PsV at MOI of 200 or mutant PsV containing the same number of reporter plasmid genomes. Infected cells were fixed at 8 or 16 h.p.i., permeabilized, and incubated with 1:100 dilutions of anti-L1 antibody and an antibody recognizing EEA1, TGN46, or VPS35. PLA was performed with Duolink reagents from Olink Biosciences according to the manufacturer's directions. Briefly, after staining with primary antibody, cells were incubated with a pair of suitable PLA antibody probes at 1:5 in a humidified chamber and processed for ligation and amplification with fluorescent substrate at 37° C. The nuclei were stained by using Abcam fluorescence mounting medium with DAPI and images were acquired by a Leica SP5 inverted fluorescence microscope. Approximately 200 nuclei in each sample were imaged. The images were processed by Fiji and quantitatively analyzed by BlobFinder software to measure total fluorescence intensity in each sample. The average fluorescence intensity per cell in each sample was normalized to the control sample as indicated in each experiment. All the experiments were done independently three times with similar results.
3×104 HeLa-S3 cells were transfected with 0.5 μg of a plasmid expressing a GFP DMT1-II fusion protein. Six hours later, medium was removed. 14 μM P16/16 or PDM/16 was added or cells were left untreated overnight. Medium was replaced with fresh medium containing same concentration of peptide, and cells were fixed and stained overnight with 1:200 dilution of anti-TGN46 antibody and then incubated with secondary antibody that recognized anti-TGN46. Cells were mounted with Abcam mounting medium with DAPI, imaged on a Leica SP5 confocal microscope, and processed with Fiji.
To determine if the cytoplasmic protrusion of L2 of PsV is inhibited by peptides, the split GFP assay was performed. 3×104 clonal HaCaT/GFP1-10NES expressing GFP1-10NES in the cytoplasm were seeded in eight-chambered glass slides and incubated with peptides for an hour prior to infection at MOI of ˜2000 with HPV16 PsV containing wild-type L2 or L2 containing inserted GFP11. Live cells were stained with Hoechst 33342, examined by a Leica SP5 confocal microscope, and processed with Fiji.
The inhibition efficiency of peptides at high MOI was tested by incubating cells with peptides at 30 μM peptide for two hours prior to infection with PsV at MOI of 2,000. At three h.p.i., cells were washed with PBS three times to remove unbound viruses and then incubated at 37° C. in the presence of 30 μM peptide. Forty-eight hours later, cells were assessed by flow cytometry to determine reporter protein expression.
The utility of potential peptide therapeutics with an intracellular site of action is limited by inefficient delivery of the peptide into cells. The L2 CPP can deliver peptides and fusion proteins into cells if added to the culture medium. We reasoned that a segment of L2 containing the CPP would deliver the adjacent RBS into the cytoplasm, sequester retromer from incoming HPV, and block HPV infection. We synthesized a 29-residue peptide, designated P16/16, that contains the RBS and CPP (RKRRKR) (SEQ ID NO: 2), where R represents arginine and K represents lysine) from HPV16 L2 (
Peptides containing the CPP from HIV Tat (P16/Tat) or HPV31 (P16/31) in place of the HPV16 CPP also markedly inhibited infection of the three HPV PsV types tested (
We used biotinylated peptides to confirm their transfer into cells and determine their intracellular location. Biotinylation did not affect anti-HPV activity (
We next identified the step of infection blocked by P16/16. Immunofluorescence studies with antibodies recognizing L1 showed that P16/16 did not inhibit virus internalization (
Finally, we tested if the L2 peptide inhibited retrograde transport of a cellular retromer cargo, DMT1-II, which contains a YLL RBS in its cytoplasmic domain required for retromer-mediated transport to the recycling endosome and TGN. We transfected cells with a plasmid expressing GFP fused to DMT1-II, and six hours later, cells were treated with P16/16 or PDM/16 or left untreated. The distribution of GFP fluorescence and anti-TGN46 antibody staining was assessed ˜20 hours later by confocal microscopy. As shown in
We describe the use of a CPP to deliver soluble peptides containing the HPV16 RBS into the cytoplasm where it sequestered retromer from L2 in the virion and inhibited endosome exit. It may be possible to increase the potency of inhibitory peptides by multimerization of the CPP or the RBS, use of an CPP or RBS from other sources, or other mutation or modification of the peptide sequence. The peptides did not display any obvious toxicity despite the importance of retromer in normal cell physiology, possibly because HPV infection is particularly dependent on retromer function. This might be the case, for example, if incoming HPV must travel via the retromer, whereas cellular cargo is constantly replenished by new synthesis as well as by retrograde transport.
We provide proof-of-principle for a new anti-viral strategy, namely inhibiting virus infection by treating cells with a peptide that enters cells and interferes with the association of the incoming virus with a cellular protein required for virus replication, in this case for proper intracellular trafficking of incoming virus. Because HPV is a localized infection of skin and mucous membranes, topical application of an entry inhibitor might be useful to prevent or limit genital HPV infection. Retromer has also been implicated in supporting the life cycle of other viruses including hepatitis C virus, influenza virus, and HIV, so agents that target retromer, including the peptides described here, may affect these viruses as well. These peptides may also be useful probes of retromer function in non-infected cells. More generally, these results identify intracellular virus trafficking as a potential therapeutic vulnerability
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.
While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/676,706, filed May 25, 2018, which is incorporated herein by reference in its entirety.
This invention was made with government support under CA016038 and AI102876 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62676706 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17057501 | Nov 2020 | US |
| Child | 18757397 | US |
