Patent Grant
11807852
Combination antiretroviral therapy can control HIV-1 replication and delay disease progression. However, despite the complete suppression of detectable viremia in many patients, viremia reemerges rapidly after interruption of treatment, consistent with the existence of a latent viral reservoir. This reservoir is thought to consist mainly of latently infected resting memory CD4+ T cells. Due to the long half-life of this reservoir (44 months), it has been estimated that its total eradication with current treatment would require over 60 years.
Latently infected cells contain replication-competent integrated HIV-1 genomes that are blocked at the transcriptional level, resulting in the absence of viral protein expression. HIV depends on both cellular and viral factors for efficient transcription of its genome, and the activity of the HIV promoter is tightly linked to the level of activation of its host cell.
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The present disclosure provides compositions and methods for reactivating latent immunodeficiency virus and/or reducing transcription of HIV integrated into the genome of an HIV-infected cell. The present disclosure provides compositions and methods for treating an immunodeficiency virus infection.
The present disclosure provides a method of reactivating latent human immunodeficiency virus (HIV) integrated into the genome of a cell infected with HIV, the method comprising contacting the cell with a Smyd2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) that reactivates latent HIV integrated into the genome of the cell. In some cases, the SMYD2 is a polypeptide comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some cases, the method comprises comprising administering at least a second agent that reactivates latent HIV. In some cases, the at least a second agent is a histone deacetylase (HDAC) inhibitor, a protein kinase C (PKC) activator, or a bromodomain inhibitor. In some cases, the HDAC inhibitor is suberoylanilidehydroxamic (SAHA), romidepsin, or sodium butyrate. In some cases, the PKC activator is prostratin, bryostatin, a chemical analog of prostratin, or a chemical analog of bryostatin. In some cases, the bromodomain inhibitor is JQ1.
The present disclosure provides a method of reducing the number of cells containing a latent human immunodeficiency virus in an individual, the method comprising administering to the individual an effective amount of a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) that reactivates latent HIV integrated into the genome of one or more cells in the individual. In some cases, said administering is effective to reduce the number of cells containing a latent human immunodeficiency virus in the individual by at least 20%. In some cases, the method comprises administering two or more agents that reactivates latent HIV integrated into the genome. In some cases, the method comprises administering a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor), and an anti-viral agent (e.g., an anti-viral agent that inhibits an immunodeficiency virus function; e.g., an anti-viral agent that inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity).
The present disclosure provides a method of treating a human immunodeficiency virus (HIV) infection in an individual, the method comprising: administering to an individual an effective amount of a first active agent, wherein the first active agent is a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) that reactivates latent HIV integrated into the genome of a cell in the individual; and administering to the individual an effective amount of a second active agent, wherein the second active agent inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity. In some cases, one or both of said administering steps is by a vaginal route of administration, by a rectal route of administration, by an oral route of administration, or by an intravenous route of administration. In some cases, the method comprises administering at least a second agent that reactivates latent HIV. In some cases, the at least a second agent is an HDAC inhibitor, a PKC activator, or a bromodomain inhibitor. In some cases, the HDAC inhibitor is SAHA, romidepsin, or sodium butyrate. In some cases, the PKC activator is prostratin, bryostatin, a chemical analog of prostratin, or a chemical analog of bryostatin. In some cases, the bromodomain inhibitor is JQ1.
The present disclosure provides a drug delivery device comprising: a) a first container comprising a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) that reactivates latent immunodeficiency virus transcription; and b) a second container comprising an agent that inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity. The first and second containers can be syringes, vials, or ampules.
In some embodiments of a method of the present disclosure, or a device of the present disclosure, the SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) is a small molecule SMYD2 inhibitor (and/or a small molecule ASH1L inhibitor, and/or a small molecule SUV420H1 inhibitor, and/or a small molecule SUV39H1 inhibitor). In some embodiments of a method of the present disclosure, or a device of the present disclosure, the SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) is an siNA, or a nucleic acid encoding an siNA. In some embodiments of a method of the present disclosure, or a device of the present disclosure, the SMYD2 inhibitor is an siNA comprising a SMYD2 shRNA nucleotide sequence set forth in
The present disclosure provides a method of identifying an agent for reactivating latent human immunodeficiency virus (HIV) integrated into the genome of a cell infected with HIV, the method comprising contacting a cell having a latent human immunodeficiency virus (HIV) integrated into the genome of the cell with a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor), and determining whether the SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) reactivates latent HIV integrated into the genome of the cell. In some cases, the SMYD2 is a polypeptide comprises an amino acid sequence having at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1. In some cases, the method comprises administering at least a second agent that reactivates latent HIV. In some cases, the at least a second agent is a histone deacetylase (HDAC) inhibitor, a protein kinase C (PKC) activator, or a bromodomain inhibitor. In some cases, the HDAC inhibitor is suberoylanilidehydroxamic (SAHA), romidepsin, or sodium butyrate. In some cases, the PKC activator is prostratin, bryostatin, a chemical analog of prostratin, or a chemical analog of bryostatin. In some cases, the bromodomain inhibitor is JQ1.
The present disclosure provides a method of identifying a candidate agent for reducing the number of cells containing a latent human immunodeficiency virus in an individual, the method comprising contacting one or more cells having a latent human immunodeficiency virus (HIV) integrated into the genome of the cells with a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor), and identifying whether the SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) reactivates latent HIV integrated into the genome of the one or more cells, wherein a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) that reactivates latent HIV integrated into the genome of the one or more cells is a candidate agent for reducing the number of cells containing a latent human immunodeficiency virus in the individual. In some cases, the method comprises contacting the one or more cells with a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor), and an anti-viral agent (e.g., an anti-viral agent that inhibits an immunodeficiency virus function; e.g., an anti-viral agent that inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity).
The present disclosure provides a method of identifying a candidate agent for treating a human immunodeficiency virus (HIV) infection in an individual, the method comprising: contacting one or more cells having a latent human immunodeficiency virus (HIV) integrated into the genome of the cells with a first active agent, wherein the first active agent is a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) that reactivates latent HIV integrated into the genome of a cell in the individual, and contacting the one or more cells with a second active agent, wherein the second active agent inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity. In some cases, the method comprises contacting the one or more cells with at least a second agent that reactivates latent HIV. In some cases, the at least a second agent is an HDAC inhibitor, a PKC activator, or a bromodomain inhibitor. In some cases, the HDAC inhibitor is SAHA, romidepsin, or sodium butyrate. In some cases, the PKC activator is prostratin, bryostatin, a chemical analog of prostratin, or a chemical analog of bryostatin. In some cases, the bromodomain inhibitor is JQ1.
As used herein, “Smyd2” or “SMYD2” (also known as SET and MYND domain containing-2 histone methyltransferase; lysine N-methyltransferase 3C; HKSM-B; KMT3C; SET and MYND domain-containing protein 2; ZMYND14; N-lysine methyltransferase SMYD2; Zinc Finger, MYND domain containing) refers to a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 9)%, or 100%, amino acid sequence identity over a contiguous stretch of from 350 amino acids to 400 amino acids, or from 400 amino acids to 433 amino acids, of the amino acid sequence depicted in
As used herein, “SMYD5” (also known as SET and MYND domain-containing protein 5) refers to a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity over a contiguous stretch of from 350 amino acids to 400 amino acids, or from 400 amino acids to 418 amino acids, of the amino acid sequence of SEQ ID NO:22. Structure and function information relating to SMYD5 is found in, e.g., Spellmon et al. (2015) Int. J. Mol. Sciences. 16:1406.
As used herein, “ASH1L” (also known as Histone-lysine N-methyltransferase ASH1L; ASH1-like protein; Absent small and homeotic disks protein 1 homolog; Lysine N-methyltransferase 2H) refers to a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity over a contiguous stretch of from 2375 amino acids to 2740 amino acids, or from 2740 amino acids to 2969 amino acids, of the amino acid sequence of SEQ ID NO: 23. Structural information relating to ASH1L is found in, e.g. An et al. (2011) J. Biol. Chem. 286: 8369.
As used herein, “SUV420H1” (also known as Histone-lysine N-methyltransferase KMT5B; lysine N-methyltransferase 5B; Lysine-specific methyltransferase 5B; Suppressor of variegation 4-20 homolog 1) refers to a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90)%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26.
As used herein, “SUV39H1” (also known as Histone-lysine N-methyltransferase SUV39H1; Histone H3-K9 methyltransferase 1; H3-K9-HMTase 1; Lysine N-methyltransferase 1A; Position-effect variegation 3-9 homolog; Suppressor of variegation 3-9 homolog 1; Su(var)3-9 homolog 1) refers to a polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence of SEQ ID NO:27 or SEQ ID NO:28. Structural information relating to SUV39H1 is found in, e.g. Wang et al. (2012) PLoS One 7(12): e52977.
The term “immunodeficiency virus” includes human immunodeficiency virus (HIV), feline immunodeficiency virus, and simian immunodeficiency virus. The term “human immunodeficiency virus” as used herein, refers to human immunodeficiency virus-1 (HIV-1); human immunodeficiency virus-2 (HIV-2); and any of a variety of HIV subtypes and quasispecies.
As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.
A “therapeutically effective amount” or “efficacious amount” refers to the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound or the cell, the disease and its severity and the age, weight, etc., of the subject to be treated.
The terms “co-administration” and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, I hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, I week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, I week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
As used herein, a “pharmaceutical composition” is meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general a “pharmaceutical composition” is sterile, and is free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal and the like. In some embodiments the composition is suitable for administration by a transdermal route, using a penetration enhancer other than dimethylsulfoxide (DMSO). In other embodiments, the pharmaceutical compositions are suitable for administration by a route other than transdermal administration. A pharmaceutical composition will in some embodiments include a subject compound and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutically acceptable excipient is other than DMSO.
As used herein, “pharmaceutically acceptable derivatives” of a compound of the invention include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced may be administered to animals or humans without substantial toxic effects and are either pharmaceutically active or are prodrugs.
A “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolanine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. 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, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a Smyd2 inhibitor” or “SMYD2 inhibitor” includes a plurality of such inhibitor and reference to “the SMYD2 polypeptide” includes reference to one or more SMYD2 polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely.” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
The present disclosure provides methods of reactivating latent HIV integrated into the genome of an HIV-infected cell and/or reducing transcription of HIV integrated into the genome of an HIV-infected cell. In some embodiments, the methods involve contacting an HIV-infected cell in which HIV is latent with an agent that inhibits methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, a SUV420H1 polypeptide or a SUV39H1 polypeptide and/or selectively reduces the level of a SMYD2 polypeptide, an ASH1L polypeptide, a SUV420H1 polypeptide or a SUV39H1 polypeptide, respectively, in the cell.
The present disclosure provides methods for reducing the reservoir of latent immunodeficiency virus in an individual, where the methods involve contacting an HIV-infected cell in which HIV is latent with an agent that inhibits methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, a SUV420H1 polypeptide or a SUV39H1 polypeptide and/or selectively reduces the level of a SMYD2 polypeptide, an ASH1L polypeptide, a SUV420H1 polypeptide or a SUV39H1 polypeptide in the cell. The present disclosure provides methods of treating an immunodeficiency virus infection in an individual, the methods generally involving co-administering to the individual an agent that reactivates latent HIV and an anti-HIV agent.
An agent that inhibits methyltransferase activity of a SMYD2 polypeptide and/or that reduces the level of a SMYD2 polypeptide in a cell, and that activates latent HIV is referred to herein as a “Smyd2 inhibitor” or a “SMYD2 inhibitor”. An agent that inhibits methyltransferase activity of an ASH1L polypeptide and/or that reduces the level of an ASH1L polypeptide in a cell, and that activates latent HIV is referred to herein as an “ASH1L inhibitor.” An agent that inhibits methyltransferase activity of a SUV420H1 polypeptide and/or that reduces the level of a SUV420H1 polypeptide in a cell, and that activates latent HIV is referred to herein as a “SUV420H1 inhibitor.” An agent that inhibits methyltransferase activity of a SUV39H1 polypeptide and/or that reduces the level of a SUV39H1 polypeptide in a cell, and that activates latent HIV is referred to herein as a “SUV39H1 inhibitor.” In some cases, a SMYD2 inhibitor suitable for use in a method of the present disclosure inhibits an enzymatic activity of SMYD2. In some cases, an ASH1L inhibitor for use in a method of the present disclosure inhibits an enzymatic activity of ASH1L. In some cases, a SUV420H1 inhibitor for use in a method of the present disclosure inhibits an enzymatic activity of SUV420H1. In some cases, a SUV39H1 inhibitor for use in a method of the present disclosure inhibits an enzymatic activity of SUV39H1. In some cases, a SMYD2 inhibitor suitable for use in a method of the present disclosure reduces the level of a SMYD2 polypeptide in a cell. In some cases, an ASH1L inhibitor suitable for use in a method of the present disclosure reduces the level of an ASH1L polypeptide in a cell. In some cases, a SUV420H1 inhibitor suitable for use in a method of the present disclosure reduces the level of a SUV420H1 polypeptide in a cell. In some cases, a SUV39H1 inhibitor suitable for use in a method of the present disclosure reduces the level of a SUV39H1 polypeptide in a cell. Regardless of the mechanism, an inhibitor suitable for use in a method of the present disclosure, e.g., a SMYD2 inhibitor, an ASH1L inhibitor, a SUV420H1 inhibitor or a SUV39H1 inhibitor, activates latent HIV in a cell harboring latent HIV.
In some cases, a suitable active agent for use in a method of the present disclosure for activating latent HIV is an agent that inhibits SMYD2 enzymatic activity, ASH1L enzymatic activity. SUV420H1 enzymatic activity or SUV39H1 enzymatic activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the enzymatic activity of the SMYD2 polypeptide, the ASH1L polypeptide, SUV420H1 polypeptide or SUV39H1 polypeptide, respectively, in the absence of the active agent. SMYD2, ASH1L, SUV420H1 or SUV39H1 enzymatic activities can be measured using any known assay for methyltransferase activity.
In some cases, a suitable active agent for use in a method of the present disclosure for activating latent HIV is an agent that reduces the level of SMYD2 polypeptide, ASH1L polypeptide, SUV420H1 polypeptide or SUV39H1 polypeptide in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the level of the SMYD2 polypeptide, the ASH1L polypeptide, the SUV420H1 polypeptide or the SUV39H1 polypeptide in the cell in the absence of the agent.
An effective amount of an active agent that inhibits methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, a SUV420H1 polypeptide or a SUV39H1 polypeptide and/or reduces the level of a SMYD2 polypeptide, an ASH1L polypeptide, a SUV420H1 polypeptide or a SUV39H1 polypeptide in a cell is an amount that reactivates latent HIV and reduces the reservoir of latent HIV in an individual by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. A “reduction in the reservoir of latent HIV” (also referred to as “reservoir of latently infected cells”) is a reduction in the number of cells in the individual that harbor a latent HIV infection. Whether the reservoir of latently infected cells is reduced can be determined using any known method, including the method described in Blankson et al. (2000) J. Infect. Disease 182(6):1636-1642.
In some cases, an effective amount of a SMYD2 inhibitor, an ASH1L inhibitor, an SUV420H1 inhibitor or an SUV39H1 inhibitor is an amount that is effective to reduce the number of cells, in a cell population, present in an individual and containing a latent human immunodeficiency virus, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The cell population can be a population of HIV-infected cells in an individual.
In some cases, a method for reducing the reservoir of latent immunodeficiency virus in an individual involves activating basal HIV-1 transcription in order to reactivate latent HIV integrated into the genome of an HIV-infected cell. The methods generally involve contacting an HIV-infected cell in which HIV is latent with an agent that induces methyltransferase activity of a SMYD5 polypeptide, and/or increases the level of a SMYD5 polypeptide in the cell. The present disclosure provides methods for reducing the reservoir of latent immunodeficiency virus in an individual, where the methods involve contacting an HIV-infected cell in which HIV is latent with an agent that induces methyltransferase activity of a SMYD5 polypeptide and/or increases the level of a SMYD5 polypeptide in the cell. The present disclosure provides methods of treating an immunodeficiency virus infection in an individual, the methods generally involving co-administering to the individual an agent that reactivates latent HIV and an anti-HIV agent.
An agent that induces methyltransferase activity of a SMYD5 polypeptide and/or that increases the level of a SMYD5 polypeptide in a cell, and that activates basal HIV-1 transcription is referred to herein as a “Smyd5 activator.” In some cases, a SMYD5 activator suitable for use in a method of the present disclosure induces an enzymatic activity of SMYD5. In some cases, a SMYD5 activator suitable for use in a method of the present disclosure increases the level of a SMYD5 polypeptide in a cell. Regardless of the mechanism, an activator suitable for use in a method of the present disclosure activates basal HIV-1 transcription in a cell harboring latent HIV.
In some cases, a suitable active agent for use in a method of the present disclosure for activating latent HIV is an agent that induces SMYD5 enzymatic, activity, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the enzymatic activity of the SMYD5 polypeptide in the absence of the active agent. Smyd5 enzymatic activity can be measured using any known assay for methyltransferase activity.
In some cases, a suitable active agent for use in a method of the present disclosure for activating basal HIV-1 transcription is an agent that increases the level of SMYD5 polypeptide in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the level of the SMYD5 polypeptide in the cell in the absence of the agent.
An effective amount of an active agent that induces methyltransferase activity of a SMYD5 polypeptide and/or increases the level of a SMYD5 polypeptide in a cell is an amount that activates basal HIV-1 transcription and reduces the reservoir of latent HIV in an individual by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.
As described above for SMYD5, an active agent that induces methyltransferase activity and/or that increases the level of one or more of the following lysine methyl transferases: MLL (comprising the amino acid sequence of any of SEQ ID NOs: 42, 43, and 44), MLL2 (comprising the amino acid sequence of SEQ ID NO: 45 or 46), MLL3 (comprising the amino acid sequence of any of SEQ ID NOs: 47, 48 and 49), MLL4 (comprising the amino acid sequence of SEQ ID NO: 50 or 51), MLL5 (comprising the amino acid sequence of any of SEQ ID NOs: 52, 53, 54 55, 56, 57, 58, and 59), SETD7/9 (comprising the amino acid sequence of SEQ ID NO: 60), SETD8 (comprising the amino acid sequence of SEQ ID NO: 61 or 62), SETDB2 (comprising the amino acid sequence of any of SEQ ID NOs: 63, 64, and 65), SETMAR (comprising the amino acid sequence of any of SEQ ID NOs: 66, 67, and 68), SMYD3 (comprising the amino acid sequence of any of SEQ ID NOs: 69, 70, and 71), and SUV420H2 (comprising the amino acid sequence of SEQ ID NO: 72 or 73), may be used in the methods described herein to reactivate latent HIV integrated into the genome of an HIV-infected cell.
SMYD5 is identified herein as an activator of HIV transcription. Accordingly, inhibitors of SMYD5, e.g., siNA or small molecule inhibitors, may find use in therapies designed to block transcription of the integrated HIV provirus. This transcriptional “shut-off” may reduce the pool of the latently infected cells by diminishing reservoir replenishment, which may accelerate the eradication of the latent reservoir. See, e.g., G. Mousseau and S. Valente, Biology, 2012, 1:668-697.
An agent that inhibits methyltransferase activity of a SMYD5 polypeptide and/or that reduces the level of a SMYD5 polypeptide in a cell is referred to herein as a “SMYD5 inhibitor.” In some cases, a SMYD5 inhibitor suitable for use in a method of the present disclosure inhibits an enzymatic activity of SMYD5. In some cases, a SMYD5 inhibitor suitable for use in a method of the present disclosure reduces the level of a SMYD5 polypeptide in a cell.
In some cases, a suitable active agent for use in a method of the present disclosure is an agent that inhibits SMYD5 enzymatic, activity, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the enzymatic activity of the SMYD5 polypeptide in the absence of the active agent. SMYD5 enzymatic activity can be measured using any known assay for methyltransferase activity.
In some cases, a suitable active agent for use in a method of the present disclosure is an agent that reduces the level of SMYD5 polypeptide in a cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, compared to the level of the SMYD5 polypeptide in the cell in the absence of the agent.
An effective amount of an active agent that inhibits methyltransferase activity of a SMYD5 polypeptide and/or reduces the level of a SMYD5 polypeptide in a cell is an amount that inhibits basal HIV-1 transcription and reduces the reservoir of latent HIV in an individual by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.
As described above for SMYD5, an active agent (e.g., siNA or small molecule inhibitors) that inhibits one or more of the following lysine methyl transferases): MLL (comprising the amino acid sequence of any of SEQ ID NOs: 42, 43, and 44), MLL2 (comprising the amino acid sequence of SEQ ID NO; 45 or 46), MLL3 (comprising the amino acid sequence of any of SEQ ID NOs: 47, 48 and 49), MLL4 (comprising the amino acid sequence of SEQ ID NO: 50 or 51), MLL5 (comprising the amino acid sequence of any of SEQ ID NOs: 52, 53, 54 55, 56, 57, 58, and 59), SETD7/9 (comprising the amino acid sequence of SEQ ID NO: 60), SETD8 (comprising the amino acid sequence of SEQ ID NO: 61 or 62), SETDB2 (comprising the amino acid sequence of any of SEQ ID NOs: 63, 64, and 65), SETMAR (comprising the amino acid sequence of any of SEQ ID NOs: 66, 67, and 68), SMYD3 (comprising the amino acid sequence of any of SEQ ID NOs: 69, 70, and 71), and SUV420H2 (comprising the amino acid sequence of SEQ ID NO: 72 or 73), may be used in the methods described herein to block transcription of the integrated HIV provirus.
In some embodiments, the present disclosure provides a screening assay designed to screen for activators or inhibitors of one or more of the lysine methyl transferases described herein. For example, methyl transferase activity can be measured, using any suitable assay, in the presence or absence of a candidate agent, e.g., a small molecule, to determine whether the candidate agent is an activator or inhibitor of the lysine methyl transferase.
Active Agents
Suitable active agents include agents that inhibit methyltransferase activity of a SMYD2 polypeptide and/or reduce the level of a SMYD2 polypeptide in a cell. Suitable active agents include SMYD2 inhibitors that reactivate latent immunodeficiency virus (e.g., HIV) in a cell.
Suitable active agents also include agents that inhibit methyltransferase activity of an ASH1L polypeptide, a SUV420H1 polypeptide and/or a SUV39H1 polypeptide and/or reduce the level of an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide in a cell. Suitable active agents include ASH1L inhibitors, SUV420H1 inhibitors and SUV39H1 inhibitors that reactivate latent immunodeficiency virus (e.g. HIV) in a cell.
Suitable active agents also include agents that reduce methytransferase activity of a SMYD5 polypeptide and/or reduce the level of a SMYD5 polypeptide in a cell. Suitable active agents include SMYD5 inhibitors that reduce basal-HIV transcription in a cell.
Small Molecule Inhibitors
In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, where the active agent is a selective SMYD2 inhibitor. In some cases, the activate agent is a small molecule inhibitor of methyltransferase activity of an ASH1L polypeptide, where the active agent is a selective ASH1L inhibitor. In some cases, the activate agent is a small molecule inhibitor of methyltransferase activity of an SUV420H1 polypeptide, where the active agent is a selective SUV420H1 inhibitor. In some cases, the activate agent is a small molecule inhibitor of methyltransferase activity of an SUV39H1 polypeptide, where the active agent is a selective SUV39H1 inhibitor. A selective SMYD2 inhibitor does not substantially inhibit a SMYD1 polypeptide, a SMYD3 polypeptide, a SMYD4 polypeptide, or a SMYD5 polypeptide, or any other methyltransferase. A selective ASH1L inhibitor does not substantially inhibit other SET domain-containing histone lysine methyltransferase or any other methyltransferase. A selective SUV420H1 inhibitor does not substantially inhibit other SET domain-containing histone lysine methyltransferase or any other methyltransferase. A selective SUV39H1 does not substantially inhibit other SET domain-containing histone lysine methyltransferase or any other methyltransferase.
In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide, and the active agent has an IC50 of from about 0.001 μM to about 100 μM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 0.001 μM to about 10 μM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 0.001 μM to about 1 μM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 0.001 μM to about 0.002 μM, from about 0.002 μM to about 0.003 μM, from about 0.003 μM to about 0.005 μM, from 0.005 μM to about 0.010 μM, from about 0.010 μM to about 0.015 μM, from about 0.015 μM to about 0.02 μM, from about 0.02 μM to about 0.05 μM, from about 0.05 μM to about 0.1 μM, from about 0.1 μM to about 0.5 μM, or from about 0.5 μM to about 1.0 μM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 1.0 μM to about 5 μM, from about 5 μM to about 10 μM, from about 10 μM to about 25 μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM, or from about 75 μM to about 100 μM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 100 μM to about 1 nM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 1 nM to about 50 nM. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD2 polypeptide, an ASH1L polypeptide, an SUV420H1 polypeptide and/or an SUV39H1 polypeptide; and the active agent has an IC50 of from about 50 nM to about 100 nM.
An example of a suitable active agent is AZ505 or a pharmaceutically acceptable derivative, e.g., salt thereof. AZ505 (N-cyclohexyl-3-((3,4-dichlorophenethyl)amino)-N-(2-((2-(5-hydroxy-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-8-yl)ethyl)amino)ethyl)propanamide bis(2,2,2-trifluoroacetate)) is a selective SMYD2 inhibitor. Ferguson et al. (2011) Structure 19:1262. AZ505 has the following structure:
In some cases, it may be desirable to administer AZ505 in combination with a cell-permeability enhancer and/or administer an AZ505 derivative which has increased cell-permeability relative to AZ505. In some cases, it may be desirable to administer AZ505 as a conjugate with a PTD or CPP as described herein.
An example of a suitable active agent is LLY-507 or a pharmaceutically acceptable derivative, e.g., salt thereof. LLY-507 is a potent inhibitor of SMYD2 with in vitro IC50 less than 15 nm, and approximately 100-fold selectivity over other methyltransferases and other non-epigenetic targets. LLY-507 has the following structure:
Another example of a suitable active agent is AZ506, also referred herein as “X1”, or a pharmaceutically acceptable derivative, e.g., salt thereof. AZ506 is a potent and selective bi-arylpiperazine, cell-permeable substrate competitive SMYD2 inhibitor with IC50 0.017 μM. AZ506 has the following structure:
An example of a suitable active agent is AZ391, also referred herein as “X2”, or a pharmaceutically acceptable derivative, e.g., salt thereof. AZ391 is a potent and selective bi-arylpiperazine substrate competitive SMYD2 inhibitor with IC50 0.062 μM. AZ391 has the following structure:
Combinations of two or more SMYD2 inhibitors can also be used in a method of the present disclosure.
An example of a suitable active agent is A-196 also known as Cyclopentyl-(6,7-dichloro-4-pyridin-4-yl-phthalazin-1-yl)-amine, or a pharmaceutically acceptable derivative, e.g., salt thereof. A-196 is a potent and selective inhibitor of SUV420H1 that inhibits the methylation of H4K20me. A-196 has the following structure:
An example of a suitable active agent is BIX-01294 also known as diazepin-quinazolin-amine derivative, or a pharmaceutically acceptable derivative, e.g., salt thereof. BIX-01294 is a SUV39H1 inhibitor that selectively impairs the generation of H3K9me2. BIX-01294 has the following structure:
An example of a suitable active agent is UNC0638 also known as 2-Cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy) quinazolin-4-amine, or a pharmaceutically acceptable derivative. e.g., salt thereof. UNC0638 is a selective inhibitor of SUV39H1. UNC0638 has the following structure:
Combinations of two or more SUV39H1 inhibitors can also be used in a method of the present disclosure.
Small Molecule Inhibitors of SMYD5
In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD5 polypeptide. In some cases, the active agent is a small molecule inhibitor of methyltransferase activity of a SMYD5 polypeptide, where the active agent is a selective SMYD5 inhibitor. A selective SMYD5 inhibitor does not substantially inhibit a SMYD1 polypeptide, a SMYD2 polypeptide, a SMYD3 polypeptide, or a SMYD4 polypeptide, or any other methyltransferase.
Inhibitors, e.g., siNA or small molecule inhibitors, may also be of interest in connection with the targeting of one or more of the additional HIV transcription activators identified in the shRNA screen described herein. For example, small molecule inhibitors of one or more of the following lysine methyl transferases: MLL (comprising the amino acid sequence of any of SEQ ID NOs: 42, 43, and 44), MLL2 (comprising the amino acid sequence of SEQ ID NO: 45 or 46), MLL3 (comprising the amino acid sequence of any of SEQ ID NOs: 47, 48 and 49), MLL4 (comprising the amino acid sequence of SEQ ID NO: 50 or 51), MLL5 (comprising the amino acid sequence of any of SEQ ID NOs: 52, 53, 54 55, 56, 57, 58, and 59), SETD7/9 (comprising the amino acid sequence of SEQ ID NO: 60), SETD8 (comprising the amino acid sequence of SEQ ID NO: 61 or 62), SETDB2 (comprising the amino acid sequence of any of SEQ ID NOs: 63, 64, and 65), SETMAR (comprising the amino acid sequence of any of SEQ ID NOs: 66, 67, and 68), SMYD3 (comprising the amino acid sequence of any of SEQ ID NOs: 69, 70, and 71), and SUV420H2 (comprising the amino acid sequence of SEQ ID NO: 72 or 73) may be used in the methods and compositions described herein. In some embodiments, such inhibitors will be selective inhibitors. Such inhibitors may be used alone or in combination with one or more inhibitors as described herein, e.g., one or more small molecule inhibitors as described herein, and/or in a combination therapy as described herein.
Nucleic Acid Inhibitors
In some cases, an active agent is a short interfering nucleic acid (siNA). The terms “short interfering nucleic acid,” “siNA,” “short interfering RNA,” “siRNA,” “shRNA,” “short interfering nucleic acid molecule,” “short interfering oligonucleotide molecule,” and “chemically-modified short interfering nucleic acid molecule” as used herein refer to any nucleic acid molecule capable of inhibiting or down regulating gene expression, for example by mediating RNA interference “RNAi” or gene silencing in a sequence-specific manner. As used herein, siNA includes short hairpin RNA (shRNA), short interfering RNA (siRNA), and the like.
A nucleic acid encoding an siNA is also contemplated for use in a method of the present disclosure, where the nucleic acid comprises a nucleotide sequence encoding the siNA. A nucleic acid encoding an siNA that reduces the level of SMYD2 polypeptide in a cell can comprise a promoter operably linked to the nucleotide sequence encoding the siNA. The nucleic acid can be present in a recombinant expression vector, e.g., a recombinant viral vector (e.g., a lentivirus-based vector; an adeno-associated virus-based vector; and the like). Suitable promoters include those that are functional in a mammalian cell, e.g., a CD4+ T cell. A suitable promoter includes, e.g., a CD4 promoter.
Non-limiting examples of suitable siNA sequences include the SMYD2 shRNA sequences depicted in
In some embodiments, siNA is produced by methods not requiring the production of dsRNA, e.g., chemical synthesis or de novo synthesis or direct synthesis. Chemically synthesized siRNA may be synthesized on a custom basis or may be synthesized on a non-custom or stock or pre-designed basis. Custom designed siRNA are routinely available from various manufactures (e.g., Ambion®, a division of Life Technologies®, Grand Island, N.Y.; Thermo Scientific®, a division of Fisher Scientific®, Pittsburgh, Pa.; Sigma-Aldrich®, St. Louis, Mo.; Qiagen®, Hilden, Germany; etc.) which provide access to various tools for the design of siRNA. Tools for the design of siNA allow for the selection of one or more siRNA nucleotide sequences based on computational programs that apply algorithms on longer input nucleotide sequences to identify candidate siNA sequences likely to be effective in producing an RNAi effect. Such algorithms can be fully automated or semi-automated, e.g., allowing for user input to guide sRNA selection. Programs applying algorithms for siNA sequence selection are available remotely on the World Wide Web, e.g., at the websites of manufacturers of chemically synthesized siNA or at the websites of independent, e.g. open source, developers or at the websites of academic institutions. Programs applying algorithms for siRNA sequence selection may also be obtained. e.g., downloaded or received on compact disk as software. Such programs are well known in the art, see e.g., Naito et al. (2004) Nucleic Acids Research, 32:W124-W129: Boudreau et al. (2013) Nucleic Acids Research, 41:e9; Mysara et al. (2011) PLoS, 6:e25642; and Iyer et al. (2007) Comput Methods Programs Biomed, 85:203-9, which are incorporated herein by reference.
Publicly available tools to facilitate design of siNAs are available in the art. See, e.g., DEQOR: Design and Quality Control of RNAi (available on the internet at http://deqor(dot)mpi-cbg(dot)de/deqor_new/input(dot)html). See also, Henschel et al. Nucleic Acids Res. 2004 Jul. 1; 32(Web Server issue):W113-20. DEQOR is a web-based program which uses a scoring system based on state-of-the-art parameters for siNA design to evaluate the inhibitory potency of siNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siNAs with high silencing potential for chemical synthesis. In addition, each siNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of siRNAs or chemically designed siRNAs.
Design of RNAi molecules, when given a target gene, is routine in the art. See also US 2005/0282188 (which is incorporated herein by reference) as well as references cited therein. See, e.g., Pushparaj et al. Clin Exp Pharmacol Physiol. 2006 May-June; 33(5-6):504-10; Lutzelberger et al. Handb Exp Pharmacol. 2006; (173):243-59; Aronin et al. Gene Ther. 2006 March; 13(6):509-16; Xie et al. Drug Discov Today. 2006 January; 11(1-2):67-73; Grunweller et al. Curr Med Chem. 2005:12(26):3143-61; and Pekaraik et al. Brain Res Bull. 2005 Dec. 15; 68(1-2):115-20. Epub 2005 Sep. 9.
Methods for design and production of siNAs to a desired target are known in the art, and their application to SMYD2 for the purposes disclosed herein will be readily apparent to the ordinarily skilled artisan, as are methods of production of siNAs having modifications (e.g., chemical modifications) to provide for, e.g., enhanced stability, bioavailability, and other properties to enhance use as therapeutics. In addition, methods for formulation and delivery of siNAs (e.g., siRNAs; shRNAs) to a subject are also well known in the art. See, e.g., US 2005/0282188; US 2005/0239731; US 2005/0234232; US 2005/0176018; US 2005/0059817; US 2005/0020525; US 2004/0192626; US 2003/0073640; US 2002/0150936; US 2002/0142980; and US2002/0120129, each of which are incorporated herein by reference.
siNA molecules can be of any of a variety of forms. For example the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, siNA can also be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary. In this embodiment, each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid or a portion thereof).
Alternatively, the siNA can be assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by a nucleic acid-based or non-nucleic acid-based linker(s). The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
The siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi. The siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (e.g., where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5′-phosphate (see for example Martinez et al., 2002, Cell, 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.
In certain embodiments, the siNA molecule contains separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the siNA molecules comprise a nucleotide sequence that is complementary to a nucleotide sequence of a target gene. In another embodiment, the siNA molecule interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
As used herein, siNA molecules need not be limited to those molecules containing only RNA, but further encompass chemically-modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules of the invention lack 2′-hydroxy (2′-OH) containing nucleotides, siNAs do not necessarily require the presence of nucleotides having a 2′-hydroxy group for mediating RNAi and as such, siNA molecules suitable for use in a method of the present disclosure optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides “siMON.”
As used herein, the term siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In some embodiments, an siNA is an siRNA. In some embodiments, an siNA is a shRNA. In some embodiments, a DNA comprising a nucleotide sequence encoding an siRNA is used. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, siNA molecules of the invention can be used to epigenetically silence a target gene (e.g., SMYD2) at the post-transcriptional level or the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siNA molecules suitable for use in a method of the present disclosure can result from siNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science. 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
siNA (e.g., siRNA; shRNA; etc.) molecules contemplated herein can comprise a duplex forming oligonucleotide (DFO) see, e.g., WO 05/019453; and US 2005/0233329, which are incorporated herein by reference), siNA molecules also contemplated herein include multifunctional siNA, (see, e.g., WO 05/019453 and US 2004/0249178).
siNA (e.g., siRNA, shRNA, etc.) molecules contemplated herein can comprise an asymmetric hairpin or asymmetric duplex. By “asymmetric hairpin” as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex with loop. For example, an asymmetric hairpin siNA molecule can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12) nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region. The asymmetric hairpin siNA molecule can also comprise a 5′-terminal phosphate group that can be chemically modified. The loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.
By “asymmetric duplex” as used herein is meant an siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complementary nucleotides to base pair with the antisense region and form a duplex. For example, an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides that are complementary to the antisense region.
Stability and/or half-life of siRNAs can be improved through chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711: Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al., supra; all of which are incorporated by reference herein, describing various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired.
For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., Intemational PCT Publication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Eamshaw and Gait, 1998, Biopolvmers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med Chem., 5, 1999-2010; each of which is hereby incorporated in their totality by reference herein). In view of such teachings, similar modifications can be used as described herein to modify the siNA nucleic acid molecules of disclosed herein so long as the ability of siNA to promote RNAi is cells is not significantly inhibited.
Short interfering nucleic acid (siNA) molecules (e.g., siRNA, shRNA, etc.) having chemical modifications that maintain or enhance activity are contemplated herein. Such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid. Accordingly, the in vitro and/or in vivo activity should not be significantly lowered. Nucleic acid molecules delivered exogenously are generally selected to be stable within cells at least for a period sufficient for transcription and/or translation of the target RNA to occur and to provide for modulation of production of the encoded mRNA and/or polypeptide so as to facilitate reduction of the level of the target gene product.
Production of RNA and DNA molecules can be accomplished synthetically and can provide for introduction of nucleotide modifications to provide for enhanced nuclease stability. (see, e.g., Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19, incorporated by reference herein. In one embodiment, nucleic acid molecules of the invention include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides, which are modified cytosine analogs which confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine within a duplex, and can provide for enhanced affinity and specificity to nucleic acid targets (see, e.g., Lin et al. 1998, J. Am. Chem. Soc., 120, 8531-8532). In another example, nucleic acid molecules can include one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (see, e.g., Wengel et al., WO 00/66604 and WO 99/14226).
siNA molecules can be provided as conjugates and/or complexes, e.g., to facilitate delivery of siNA molecules into a cell. Exemplary conjugates and/or complexes include those composed of an siNA and a small molecule, lipid, cholesterol, phospholipid, nucleoside, antibody, toxin, negatively charged polymer (e.g., protein, peptide, hormone, carbohydrate, polyethylene glycol, or polyamine). In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds can improve delivery and/or localization of nucleic acid molecules into cells in the presence or absence of serum (see, e.g., U.S. Pat. No. 5,854,038). Conjugates of the siNA molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
Nucleic Acid Modifications
In some embodiments, a SMYD2 inhibitor (e.g., a dsRNA, a siNA, etc.) has one or more modifications, e.g., a base modification, a backbone modification, etc., to provide the nucleic acid with an enhanced feature (e.g., improved stability). A nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, the 3′, or the 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Suitable nucleic acid modifications include, but are not limited to: 2′-O-methyl modified nucleotides, 2′ Fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate (PS) linkages, and a 5′ cap (e.g., a 7-methylguanylate cap (m7G)). Additional details and additional modifications are described below.
A 2′-O-Methyl modified nucleotide (also referred to as 2′-O-Methyl RNA) is a naturally occurring modification of RNA found in tRNA and other small RNAs that arises as a post-transcriptional modification. Oligonucleotides can be directly synthesized that contain 2′-O-Methyl RNA. This modification increases the melting temperature (Tm) of RNA:RNA duplexes but results in only small changes in RNA:DNA stability. It is stabile with respect to attack by single-stranded ribonucleases and is typically 5 to 10-fold less susceptible to DNases than DNA. It is commonly used in antisense oligos as a means to increase stability and binding affinity to the target message.
2′ Fluoro modified nucleotides (e.g., 2′ Fluoro bases) have a fluorine modified ribose which increases binding affinity (Tm) and also confers some relative nuclease resistance when compared to native RNA These modifications are commonly employed in ribozymes and siNAs to improve stability in serum or other biological fluids.
Locked nucleic acid (LNA) bases have a modification to the ribose backbone that locks the base in the C3′-endo position, which favors RNA A-type helix duplex geometry. This modification significantly increases Tm and is also very nuclease resistant. Multiple LNA insertions can be placed in an oligonucleotide (“oligo”) at any position except the 3′-end. Due to the large increase in Tm conferred by LNAs, they also can cause an increase in primer dimer formation as well as self-hairpin formation. In some cases, the number of LNAs incorporated into a single oligo is 10 bases or less.
The phosphorothioate (PS) bond (i.e., a phosphorothioate linkage) substitutes a sulfur atom for a non-bridging oxygen in the phosphate backbone of a nucleic acid (e.g., an oligo). This modification renders the internucleotide linkage resistant to nuclease degradation. Phosphorothioate bonds can be introduced between the last 3-5 nucleotides at the 5′- or 3′-end of the oligo to inhibit exonuclease degradation. Including phosphorothioate bonds within the oligo (e.g., throughout the entire oligo) can help reduce attack by endonucleases as well.
In some embodiments, a subject siNA (e.g., siNA, shRNA, etc.) has one or more nucleotides that are 2′-O-Methyl modified nucleotides. In some embodiments, a subject siNA (e.g., a dsRNA, a siNA, a shRNA, etc.) has one or more 2′ Fluoro modified nucleotides. In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, a shRNA, etc.) has one or more LNA bases. In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, a shRNA, etc.) has one or more nucleotides that are linked by a phosphorothioate bond (i.e., the subject nucleic acid has one or more phosphorothioate linkages). In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, an shRNA, etc.) has a 5′ cap (e.g., a 7-methylguanylate cap (m7G)). In some embodiments, a subject nucleic acid (e.g., a dsRNA, a siNA, a shRNA, etc.) has a combination of modified nucleotides. For example, a subject nucleic acid (e.g., a dsRNA, a siNA, etc.) can have a 5′ cap (e.g., a 7-methylguanylate cap (m7G)) in addition to having one or more nucleotides with other modifications (e.g., a 2′-O-Methyl nucleotide and/or a 2′ Fluoro modified nucleotide and/or a LNA base and/or a phosphorothioate linkage).
Modified Backbones and Modified Internucleoside Linkages
Examples of suitable nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included.
In some embodiments, a subject siNA comprises one or more phosphorothioate and/or heteroatom internucleoside linkages, in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— (known as a methylene (methylimino) or MMI backbone), —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —O—N(CH3)—CH2—CH2— (wherein the native phosphodiester internucleotide linkage is represented as —O—P(═O)(OH)—O—CH2—). MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Suitable amide internucleoside linkages are disclosed in U.S. Pat. No. 5,602,240.
Also suitable are nucleic acids having morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506. For example, in some embodiments, a subject nucleic acid comprises a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.
Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones, methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Mimetics
A subject siNA can be a nucleic acid mimetic. The term “mimetic” as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
One polynucleotide mimetic that has been reported to have excellent hybridization properties is a peptide nucleic acid (PNA). The backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.
Another class of polynucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups has been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry. 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.
A further class of polynucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general, the incorporation of CeNA monomers into a DNA chain increases the stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.
A further modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugar ring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (—CH2—), group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10° C.), stability towards 3′-exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (e.g., Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).
The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (e.g., Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226, as well as U.S. Patent Publication Nos. 20120165514, 20100216983, 20090041809, 20060117410, 20040014959, 20020094555, and 20020086998.
Modified Sugar Moieties
A subject siNA can also include one or more substituted sugar moieties.
Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A suitable modification includes 2′-methoxyethoxy (2′-O—CH2 CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitable modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH3)2.
Other suitable sugar substituent groups include methoxy (—O—CH3), aminopropoxy (—O CH2 CH2 CH2NH2), allyl (—CH2—CH═CH2), —O-allyl (—O—CH2—CH═CH2) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Base Modifications and Substitutions
A subject siNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3′,2′:4,5)pyrrolo(2,3-d)pyrimidin-2-one).
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are useful for increasing the binding affinity of an oligomeric compound. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable base substitutions, e.g., when combined with 2′-O-methoxyethyl sugar modifications.
Conjugates
Another possible modification of a subject siNA involves chemically linking to the polynucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Suitable conjugate groups include, but are not limited to, cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of a subject siNA.
Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Boorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acid & Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654: Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937.
A conjugate may include a “Protein Transduction Domain” or PTD (also known as a CPP—cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the 3′ terminus of an exogenous polynucleotide (e.g., a siNA). In some embodiments, a PTD is covalently linked to the 5′ terminus of an exogenous polynucleotide (e.g., a siNA). Exemplary PTDs include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO: 11)); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:12): Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:13); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:14); and RQIKIWFQNRRMKWKK (SEQ ID NO:15). Exemplary PTDs include but are not limited to, YGRKKRRQRRR (SEQ ID NO:11), RKKRRQRRR (SEQ ID NO:16): an arginine homopolymer of from 3 arginine residues to 50 arginine residues; Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:11); RKKRRQRR (SEQ ID NO:17): YARAAARQARA (SEQ ID NO:18); THRLPRRRRRR (SEQ ID NO:19), and GGRRARRRRRR (SEQ ID NO:20). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.
Combination Therapy
The present disclosure provides combination therapy for treating an immunodeficiency virus infection in an individual.
Combination Therapy—Two or More Agents that Reactivate Latent HIV
In some embodiments, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves administering to the individual an effective amount of two or more agents that activate immunodeficiency virus transcription. In some cases, the two or more agents act synergistically to reactivate latent immunodeficiency virus.
In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of a SMYD2 inhibitor, an ASH1L inhibitor, an SUV420H1 inhibitor and/or a SUV39H1 inhibitor that activates immunodeficiency virus transcription; and b) administering to the individual an effective amount of a second agent that activates latent immunodeficiency virus transcription.
Suitable second agents that activate latent immunodeficiency virus transcription include, e.g., a bromodomain inhibitor; a protein kinase C (PKC) activator, such as prostratin, bryostatin, a chemical analog of prostratin, a chemical analog of bryostatin, and the like; a histone deacetylase (HDAC) inhibitor such as suberoylanilidehydroxamic (SAHA), romidepsin, sodium butyrate, and the like.
Bromodomain inhibitors suitable for use include, e.g., JQ1, which has the following structure:
Suitable bromodomain inhibitors include compounds of formula I:
R1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, and acyl;
R2 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, and acyl;
R3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, and acyl;
R4a is selected from hydrogen, C1-C3 alkyl, C5-C10 alkyl, and substituted alkyl;
R5 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, hydroxy, alkoxy, substituted alkoxy, acyloxy, thiol, acyl, amino, substituted amino, aminoacyl, acylamino, azido, carboxyl, carboxylalkyl, cyano, halogen, and nitro;
and salts or solvates or stereoisomers thereof.
In formula I, R1 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, and acyl. In certain instances, R1 is hydrogen. In certain instances, R1 is alkyl or substituted alkyl. In certain instances. R1 is alkyl, such as C1-C6 alkyl, including C1-C3 alkyl. In certain instances, R1 is methyl, ethyl, n-propyl, or isopropyl. In certain instances, R1 is methyl. In certain instances, R1 is alkenyl or substituted alkenyl. In certain instances, R1 is selected from alkynyl or substituted alkynyl. In certain instances, R1 is alkoxy or substituted alkoxy. In certain instances, R1 is acyl.
In formula I, R2 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, and acyl. In certain instances, R2 is hydrogen. In certain instances, R2 is alkyl or substituted alkyl. In certain instances, R2 is alkyl, such as C1-C6 alkyl, including C1-C3 alkyl. In certain instances, R2 is methyl, ethyl, n-propyl, or isopropyl. In certain instances, R2 is methyl. In certain instances, R2 is alkenyl or substituted alkenyl. In certain instances, R2 is selected from alkynyl or substituted alkynyl. In certain instances. R2 is alkoxy or substituted alkoxy. In certain instances, R2 is acyl.
In formula I, R3 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, and acyl. In certain instances, R3 is hydrogen. In certain instances, R1 is alkyl or substituted alkyl. In certain instances, R3 is alkyl, such as C1-C6 alkyl, including C1-C3 alkyl. In certain instances, R3 is methyl, ethyl, n-propyl, or isopropyl. In certain instances, R3 is methyl. In certain instances, R3 is alkenyl or substituted alkenyl. In certain instances, R1 is selected from alkynyl or substituted alkynyl. In certain instances, R3 is alkoxy or substituted alkoxy. In certain instances, R3 is acyl.
In formula I, R4a is selected from hydrogen, C1-C3 alkyl, C5-C10 alkyl, and substituted alkyl. In certain instances, R4a is hydrogen. In certain instances, R4a is C1-C3 alkyl. In certain instances, R4a is C5-C10 alkyl. In certain instances, R4a is substituted alkyl. In certain instances, R4a is methyl, ethyl, n-propyl, or isopropyl. In certain instances, R41 is methyl.
In formula I, R5 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, hydroxy, alkoxy, substituted alkoxy, acyloxy, thiol, acyl, amino, substituted amino, aminoacyl, acylamino, azido, carboxyl, carboxylalkyl, cyano, halogen, and nitro.
In certain instances, R5 is hydrogen. In certain instances, R5 is alkyl or substituted alkyl. In certain instances, R5 is alkenyl or substituted alkenyl. In certain instances, R5 is alkynyl or substituted alkynyl. In certain instances, R5 is hydroxy, alkoxy, substituted alkoxy, or acyloxy. In certain instances, R5 is thiol. In certain instances, R5 is acyl. In certain instances, R5 is amino, substituted amino, aminoacyl, acylamino, or azido. In certain instances, R5 is carboxyl or carboxylalkyl. In certain instances, R5 is cyano. In certain instances, R5 is nitro. In certain instances, R5 is halogen. In certain instances, R5 is fluoro. In certain instances, R5 is chloro. In certain instances, R5 is bromo.
In certain instances, formula I is the following formula:
A particular compound of interest, and salts or solvates or stereoisomers thereof, includes:
(Methyl 2-((6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate).
Suitable HDAC inhibitors include hydroxamic acids (e.g., vorinostat (suberoylanilide hydroxamic acid, SAHA, Archin et al., AIDS Res Hum Retroviruses, 25(2): 207-12, 2009; Contreras et al. J Biol Chem, 284:6782-9, 2009), belinostat (PXD101), LAQ824; and panobinostat (LBH589); and benzamides (e.g., entinostat (MS-275), C1994; and mocetinostat (MGCD0103). Suitable HDAC inhibitors include butyric acid (including sodium butyrate and other salt forms), Valproic acid (including Mg valproate and other salt forms), suberoylanilide hydroxamic acid (SAHA), Vorinostat, Romidepsin (trade name Istodax), Panobinostat (LBH589), Belinostat (PXD101). Mocetinostat (MGCD0103), PCI-24781, Entinostat (MS-275), SB939, Resminostat (4SC-201); Givinostat (ITF2357), CUDC-101, AR-42, CHR-2845, CHR-3996, 4SC-202, sulforaphane, BML-210, M344, CI-994; CI-994 (Tacedinaline); BML-210; M344; MGCDO103 (Mocetinostat); and Tubastatin A. Additional suitable HDAC inhibitors are described in U.S. Pat. No. 7,399,787.
Suitable bryostatins include bryostatin-1; a bryostatin analog as described in U.S. Pat. No. 6,624,189: bryostatin-2, a bryostatin analog as described in U.S. Pat. No. 7,256,286; a bryostatin analog described in U.S. Patent Publication No. 20090270492, a bryostatin analog described in WO 2013/165592: etc.
In some embodiments, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of two or more agents that activate immunodeficiency virus transcription; and b) administering to the individual an effective amount of an agent that inhibits an immunodeficiency virus function. The immunodeficiency virus function can be selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.
In some embodiments, the co-administration of compounds results in synergism, and the combination is therefore a synergistic combination. As used herein, a “synergistic combination” or a “synergistic amount” of (i) a SMYD2 inhibitor that activates immunodeficiency virus transcription; and (ii) a second agent that activates immunodeficiency virus transcription is an amount that is more effective in activating immunodeficiency virus transcription when co-administered than the incremental increase that could be predicted or expected from a merely additive combination of (i) and (ii) when each is administered at the same dosage alone (not co-administered).
In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ505; and b) administering to the individual an effective amount of JQ1. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ505; and b) administering to the individual an effective amount of SAHA. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ505; and b) administering to the individual an effective amount of bryostatin or a bryostatin analog. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ505; and b) administering to the individual an effective amount of an HDAC inhibitor. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ505; and b) administering to the individual an effective amount of prostratin or a prostratin analog.
In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ506 and/or AZ391 (or another suitable methyltransferase inhibitor as described herein), or a pharmaceutically acceptable derivative, e.g., salt thereof, and b) administering to the individual an effective amount of JQ1. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ506 and/or AZ391 (or another suitable methyltransferase inhibitor as described herein), or a pharmaceutically acceptable derivative, e.g., salt thereof; and b) administering to the individual an effective amount of SAHA. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ506 and/or AZ391 (or another suitable methyltransferase inhibitor as described herein), or a pharmaceutically acceptable derivative, e.g., salt thereof; and b) administering to the individual an effective amount of bryostatin or a bryostatin analog. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ506 and/or AZ391 (or another suitable methyltransferase inhibitor as described herein), or a pharmaceutically acceptable derivative, e.g., salt thereof; and b) administering to the individual an effective amount of an HDAC inhibitor. In some cases, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of AZ506 and/or AZ391 (or another suitable methyltransferase inhibitor as described herein), or a pharmaceutically acceptable derivative, e.g., salt thereof; and b) administering to the individual an effective amount of prostratin or a prostratin analog.
Combination Therapy—SMYD2 Inhibitor (and/or ASH1L Inhibitor and/or SUV420H1 Inhibitor and/or SUV39H1 Inhibitor)+Anti-Viral Agent
In some embodiments, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of a SMYD2 inhibitor (and/or ASH1L inhibitor and/or SUV420H1 inhibitor and/or SUV39H1 inhibitor) that activates immunodeficiency virus transcription; and b) administering to the individual an effective amount of an agent that inhibits an immunodeficiency virus function. The immunodeficiency virus function can be selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.
In some embodiments, a method of the present disclosure of treating an immunodeficiency virus infection in an individual in need thereof involves: a) administering to the individual an effective amount of an agent that inhibits SMYD2 enzymatic activity (and/or ASH1L enzymatic activity and/or SUV420H1 enzymatic activity and/or SUV39H1 enzymatic activity) and/or reduces the level of SMYD2 polypeptide (and/or ASH1L polypeptide and/or SUV420H1 polypeptide and/or SUV39H1 polypeptide) in a cell, and that activates immunodeficiency virus transcription; and b) administering to the individual an effective amount of an agent that inhibits an immunodeficiency virus function. The immunodeficiency virus function can be selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.
In some embodiments, a compound that is a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) (e.g., an agent that inhibits SMYD2 enzymatic activity and/or reduces the level of SMYD2 polypeptide in a cell) and that activates immunodeficiency virus transcription is administered in combination therapy (i.e., co-administered) with: 1) one or more nucleoside reverse transcriptase inhibitors (e.g., Combivir, Epivir, Hivid, Retrovir, Videx, Zerit, Ziagen, etc.); 2) one or more non-nucleoside reverse transcriptase inhibitors (e.g., Rescriptor, Sustiva, Viramune, etc.); 3) one or more protease inhibitors (e.g., Agenerase, Crixivan, Fortovase, Invirase, Kaletra, Norvir, Viracept, etc.); 4) an anti-HIV agent such as a protease inhibitor and a nucleoside reverse transcriptase inhibitor: 5) an anti-HIV agent such as a protease inhibitor, a nucleoside reverse transcriptase inhibitor, and a non-nucleoside reverse transcriptase inhibitor; 6) an anti-HIV agent such as a protease inhibitor and a non-nucleoside reverse transcriptase inhibitor, and/or 7) an anti-viral (e.g., HIV) agent such as a protein kinase C (PKC) activator (e.g., prostratin). Other combinations of an effective amount of a SMYD2 inhibitor with one or more anti-HIV agents, such as one or more of a protease inhibitor, a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, and a protein kinase C (PKC) activator are contemplated.
A PKC activator (e.g., prostratin ((1aR,1bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl)) can be administered in a separate formulation from a SMYD2 inhibitor. A PKC activator can be co-formulated with a SMYD2 inhibitor, and the co-formulation administered to an individual.
In some embodiments, the co-administration of compounds results in synergism, and the combination is therefore a synergistic combination. As used herein, a “synergistic combination” or a “synergistic amount” of (i) a SMYD2 inhibitor that activates immunodeficiency virus transcription and (ii) an anti-viral agent (e.g., a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a protease inhibitor, an anti-HIV agent, a protein kinase C (PKC) activator, etc.) is an amount that is more effective in reducing immunodeficiency virus load when co-administered than the incremental increase that could be predicted or expected from a merely additive combination of (i) and (ii) when each is administered at the same dosage alone (not co-administered). As used herein, a “synergistic combination” or a “synergistic amount” of (i) a SMYD2 inhibitor that activates immunodeficiency virus transcription and (ii) a second agent that activates latent immunodeficiency virus transcription, is an amount that is more effective in reactivating latent immunodeficiency virus transcription when co-administered than the incremental increase that could be predicted or expected from a merely additive combination of (i) and (ii) when each is administered at the same dosage alone (not co-administered).
Any of a variety of methods can be used to determine whether a treatment method is effective. For example, methods of determining whether the methods of the present disclosure are effective in reducing immunodeficiency virus (e.g., HIV) viral load, and/or treating an immunodeficiency virus (e.g., HIV) infection, are any known test for indicia of immunodeficiency virus (e.g., HIV) infection, including, but not limited to, measuring viral load, e.g., by measuring the amount of immunodeficiency virus (e.g., HIV) in a biological sample, e.g., using a polymerase chain reaction (PCR) with primers specific for an immunodeficiency virus (e.g., HIV) polynucleotide sequence; detecting and/or measuring a polypeptide encoded by an immunodeficiency virus (e.g., HIV), e.g., p24, gp120, reverse transcriptase, using, e.g., an immunological assay such as an enzyme-linked immunosorbent assay (ELISA) with an antibody specific for the polypeptide; and measuring the CD4+ T cell count in the individual.
Formulations, Dosages, and Routes of Administration
In general, an active agent (e.g., a SMYD2 inhibitor) is prepared in a pharmaceutically acceptable composition(s) for delivery to a host. In the context of reducing immunodeficiency virus transcription, the terms “active agent,” “drug,” “agent,” “therapeutic agent,” and the like are used interchangeably herein to refer to an agent that is a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) and that activates latent immunodeficiency virus transcription.
Pharmaceutically acceptable carriers suitable for use with active agents (and optionally one or more additional therapeutic agents) may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, and microparticles, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. A composition comprising an active agent (and optionally one or more additional therapeutic agent) may also be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.
Formulations
An active agent is administered to an individual in need thereof in a formulation with a pharmaceutically acceptable excipient(s). A wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington. The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7h ed., Lippincott, Williams. & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. For the purposes of the following description of formulations, “active agent” includes an active agent as described above, and optionally one or more additional therapeutic agent.
In a subject method, an active agent may be administered to the host using any convenient means capable of resulting in the desired degree of reduction of immunodeficiency virus transcription. Thus, an active agent can be incorporated into a variety of formulations for therapeutic administration. For example, an active agent can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In an exemplary embodiment, an active agent is formulated as a gel, as a solution, or in some other form suitable for intravaginal administration. In a further exemplary embodiment, an active agent is formulated as a gel, as a solution, or in some other form suitable for rectal (e.g., intrarectal) administration.
In pharmaceutical dosage forms, an active agent may be administered in the form of its pharmaceutically acceptable salts, or it may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
In some embodiments, an active is formulated in an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers varying in strengths from about 5 mM to about 100 mM. In some embodiments, the aqueous buffer includes reagents that provide for an isotonic solution. Such reagents include, but are not limited to, sodium chloride; and sugars e.g., mannitol, dextrose, sucrose, and the like. In some embodiments, the aqueous buffer further includes a non-ionic surfactant such as polysorbate 20 or 80. Optionally the formulations may further include a preservative. Suitable preservatives include, but are not limited to, a benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, and the like. In many cases, the formulation is stored at about 4° C. Formulations may also be lyophilized, in which case they generally include cryoprotectants such as sucrose, trehalose, lactose, maltose, mannitol, and the like. Lyophilized formulations can be stored over extended periods of time, even at ambient temperatures.
For oral preparations, an active agent can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
An active agent can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
An active agent can be utilized in aerosol formulation to be administered via inhalation. An active agent can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, an active agent can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. An active agent can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more active agents. Similarly, unit dosage forms for injection or intravenous administration may comprise the active agent(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
Unit dosage forms for intravaginal or intrarectal administration such as syrups, elixirs, gels, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet, unit gel volume, or suppository, contains a predetermined amount of the composition containing one or more active agents.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of an active agent, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a given active agent will depend in part on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
Other modes of administration will also find use with a method of the present disclosure. For instance, an active agent can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), e.g. about 1% to about 2%.
An active agent can be administered in an injectable formulation. Typically, injectable compositions are prepared as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.
An active agent will in some embodiments be formulated for vaginal delivery. A subject formulation for intravaginal administration comprises an active agent formulated as an intravaginal bioadhesive tablet, intravaginal bioadhesive microparticle, intravaginal cream, intravaginal lotion, intravaginal foam, intravaginal ointment, intravaginal paste, intravaginal solution, or intravaginal gel.
An active agent will in some embodiments be formulated for rectal delivery. A subject formulation for intrarectal administration comprises an active agent formulated as an intrarectal bioadhesive tablet, intrarectal bioadhesive microparticle, intrarectal cream, intrarectal lotion, intrarectal foam, intrarectal ointment, intrarectal paste, intrarectal solution, or intrarectal gel.
A subject formulation comprising an active agent includes one or more of an excipient (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, poly(ethylene glycol), sucrose or starch), a disintegrator (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or orange powder), a preservative (e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben), a stabilizer (e.g., citric acid, sodium citrate or acetic acid), a suspending agent (e.g., methylcellulose, polyvinylpyrrolidone or aluminum stearate), a dispersing agent (e.g., hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax (e.g., cocoa butter, white petrolatum or polyethylene glycol).
Tablets comprising an active agent may be coated with a suitable film-forming agent, e.g., hydroxypropylmethyl cellulose, hydroxypropyl cellulose or ethyl cellulose, to which a suitable excipient may optionally be added, e.g., a softener such as glycerol, propylene glycol, diethylphthalate, or glycerol triacetate; a filler such as sucrose, sorbitol, xylitol, glucose, or lactose; a colorant such as titanium hydroxide; and the like.
Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Dosages
Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range of an active agent is one which provides up to about 1 mg to about 1000 mg, e.g., from about 1 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 200 mg, from about 200 mg to about 250 mg, from about 250 mg to about 500 mg, or from about 500 mg to about 1000 mg of an active agent can be administered in a single dose.
Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
In some embodiments, a single dose of an active agent is administered. In other embodiments, multiple doses of an active agent are administered. Where multiple doses are administered over a period of time, an active agent is administered twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time. For example, an active agent is administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, an active agent is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors.
Where two different active agents are administered, a first active agent and a second active agent can be administered in separate formulations. A first active agent and a second active agent can be administered substantially simultaneously, or within about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 72 hours, about 4 days, about 7 days, or about 2 weeks of one another.
Routes of Administration
An active agent is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, transdermal, subcutaneous, intradermal, topical application, intravenous, vaginal, nasal, and other parenteral routes of administration. In some embodiments, an active agent is administered via an intravaginal route of administration. In other embodiments, an active agent is administered via an intrarectal route of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses.
An active agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.
Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, vaginal, transdermal, subcutaneous, intramuscular, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
An active agent can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.
By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as the number of viral particles per unit blood. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition.
A variety of hosts (wherein the term “host” is used interchangeably herein with the terms “subject” and “patient”) are treatable according to the subject methods. Generally such hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, and primates (e.g., humans, chimpanzees, and monkeys), that are susceptible to immunodeficiency virus (e.g., HIV) infection. In many embodiments, the hosts will be humans.
Kits, Containers, Devices, Delivery Systems
Kits with unit doses of the active agent, e.g. in oral, vaginal, rectal, transdermal, or injectable doses (e.g., for intramuscular, intravenous, or subcutaneous injection), are provided. In such kits, in addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in treating an immunodeficiency virus (e.g., an HIV) infection. Suitable active agents and unit doses are those described herein above.
In many embodiments, a subject kit will further include instructions for practicing the subject methods or means for obtaining the same (e.g., a website URL directing the user to a webpage which provides the instructions), where these instructions are typically printed on a substrate, which substrate may be one or more of: a package insert, the packaging, formulation containers, and the like.
In some embodiments, a subject kit includes one or more components or features that increase patient compliance, e.g., a component or system to aid the patient in remembering to take the active agent at the appropriate time or interval. Such components include, but are not limited to, a calendaring system to aid the patient in remembering to take the active agent at the appropriate time or interval.
The present invention provides a delivery system comprising an active agent (a SMYD2 inhibitor; optionally also one or more additional therapeutic agents). In some embodiments, the delivery system is a delivery system that provides for injection of a formulation comprising an active agent subcutaneously, intravenously, or intramuscularly. In other embodiments, the delivery system is a vaginal or rectal delivery system.
In some embodiments, an active agent is packaged for oral administration. The present invention provides a packaging unit comprising daily dosage units of an active agent. For example, the packaging unit is in some embodiments a conventional blister pack or any other form that includes tablets, pills, and the like. The blister pack will contain the appropriate number of unit dosage forms, in a sealed blister pack with a cardboard, paperboard, foil, or plastic backing, and enclosed in a suitable cover. Each blister container may be numbered or otherwise labeled, e.g., starting with day 1.
In some embodiments, a delivery system of the present disclosure comprises an injection device. Exemplary, non-limiting drug delivery devices include injections devices, such as pen injectors, and needle/syringe devices. In some embodiments, the invention provides an injection delivery device that is pre-loaded with a formulation comprising an effective amount of a SMYD2 inhibitor. For example, a subject delivery device comprises an injection device pre-loaded with a single dose of a SMYD2 inhibitor. A injection device can be re-usable or disposable.
Pen injectors are well known in the art. Exemplary devices which can be adapted for use in the present methods are any of a variety of pen injectors from Becton Dickinson. e.g., BD™ Pen, BD™ Pen II, BD™ Auto-Injector; a pen injector from Innoject. Inc.: any of the medication delivery pen devices discussed in U.S. Pat. Nos. 5,728,074, 6,096,010, 6,146,361, 6,248,095, 6,277,099, and 6,221,053; and the like. The medication delivery pen can be disposable, or reusable and refillable.
The present invention provides a delivery system for vaginal or rectal delivery of an active agent to the vagina or rectum of an individual. The delivery system comprises a device for insertion into the vagina or rectum. In some embodiments, the delivery system comprises an applicator for delivery of a formulation into the vagina or rectum, and a container that contains a formulation comprising an active agent. In these embodiments, the container (e.g., a tube) is adapted for delivering a formulation into the applicator. In other embodiments, the delivery system comprises a device that is inserted into the vagina or rectum, which device includes an active agent. For example, the device is coated with, impregnated with, or otherwise contains a formulation comprising the active agent.
In some embodiments, the vaginal or rectal delivery system is a tampon or tampon-like device that comprises a subject formulation. Drug delivery tampons are known in the art, and any such tampon can be used in conjunction with a subject drug delivery system. Drug delivery tampons are described in, e.g., U.S. Pat. No. 6,086,909. If a tampon or tampon-like device is used, there are numerous methods by which an active agent can be incorporated into the device. For example, the drug can be incorporated into a gel-like bioadhesive reservoir in the tip of the device. Alternatively, the drug can be in the form of a powdered material positioned at the tip of the tampon. The drug can also be absorbed into fibers at the tip of the tampon, for example, by dissolving the drug in a pharmaceutically acceptable carrier and absorbing the drug solution into the tampon fibers. The drug can also be dissolved in a coating material which is applied to the tip of the tampon. Alternatively, the drug can be incorporated into an insertable suppository which is placed in association with the tip of the tampon.
In other embodiments, the drug delivery device is a vaginal or rectal ring. Vaginal or rectal rings usually consist of an inert elastomer ring coated by another layer of elastomer containing an active agent to be delivered. The rings can be easily inserted, left in place for the desired period of time (e.g., up to 7 days), then removed by the user. The ring can optionally include a third, outer, rate-controlling elastomer laver which contains no drug. Optionally, the third ring can contain a second drug for a dual release ring. The drug can be incorporated into polyethylene glycol throughout the silicone elastomer ring to act as a reservoir for drug to be delivered.
In other embodiments, a subject vaginal or rectal delivery system is a vaginal or rectal sponge. The active agent is incorporated into a silicone matrix which is coated onto a cylindrical drug-free polyurethane sponge, as described in the literature.
Pessaries, tablets, and suppositories are other examples of drug delivery systems which can be used, e.g., in carrying out a method of the present disclosure. These systems have been described extensively in the literature.
Bioadhesive microparticles constitute still another drug delivery system suitable for use in the present invention. This system is a multi-phase liquid or semi-solid preparation which does not seep from the vagina or rectum as do many suppository formulations. The substances cling to the wall of the vagina or rectum and release the drug over a period of time. Many of these systems were designed for nasal use but can be used in the vagina or rectum as well (e.g. U.S. Pat. No. 4,756,907). The system may comprise microspheres with an active agent; and a surfactant for enhancing uptake of the drug. The microparticles have a diameter of 10-100 μm and can be prepared from starch, gelatin, albumin, collagen, or dextran.
Another system is a container comprising a subject formulation (e.g., a tube) that is adapted for use with an applicator. The active agent is incorporated into creams, lotions, foams, paste, ointments, and gels which can be applied to the vagina or rectum using an applicator. Processes for preparing pharmaceuticals in cream, lotion, foam, paste, ointment and gel formats can be found throughout the literature. An example of a suitable system is a standard fragrance free lotion formulation containing glycerol, ceramides, mineral oil, petrolatum, parabens, fragrance and water such as the product sold under the trademark JERGENS™ (Andrew Jergens Co., Cincinnati, Ohio). Suitable nontoxic pharmaceutically acceptable systems for use in the compositions of the present invention will be apparent to those skilled in the art of pharmaceutical formulations and examples are described in Remington's Pharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., 1995. The choice of suitable carriers will depend on the exact nature of the particular vaginal or rectal dosage form desired. e.g., whether the active ingredient(s) is/are to be formulated into a cream, lotion, foam, ointment, paste, solution, or gel, as well as on the identity of the active ingredient(s). Other suitable delivery devices are those described in U.S. Pat. No. 6,476,079.
Combination Therapy
In some embodiments, a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) is administered in combination therapy with one or more additional therapeutic agents. Suitable additional therapeutic agents include agents that inhibit one or more functions of an immunodeficiency virus: agents that treat or ameliorate a symptom of an immunodeficiency virus infection; agents that treat an infection that occurs secondary to an immunodeficiency virus infection; and the like. As noted above, suitable additional therapeutic agents include agents (other than a SMYD2 inhibitor) that reactivate latent immunodeficiency virus.
Therapeutic agents include, e.g., beta-lactam antibiotics, tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone, hydrocortisone, betamethasone, dexamethasone, fluocortolone, prednisolone, triamcinolone, indomethacin, sulindac, acyclovir, amantadine, rimantadine, recombinant soluble CD4 (rsCD4), anti-receptor antibodies (e.g., for rhinoviruses), nevirapine, cidofovir (Vistide™), trisodium phosphonoformate (Foscamet™), famcyclovir, pencyclovir, valacyclovir, nucleic acid/replication inhibitors, interferon, zidovudine (AZT, Retrovir™), didanosine (dideoxyinosine, ddI, Videx™), stavudine (d4T, Zerit™), zalcitabine (dideoxycytosine, ddC, Hivid™), nevirapine (Viramune™), lamivudine (Epivir™, 3TC), protease inhibitors, saquinavir (Invirase™, Fortovase™), ritonavir (Norvir™), nelfinavir (Viracept™), efavirenz (Sustivar™), abacavir (Ziagen™), amprenavir (Agenerase™) indinavir (Crixivan™), ganciclovir, AzDU, delavirdine (Rescriptor™), kaletra, trizivir, rifampin, clathiromycin, erythropoietin, colony stimulating factors (G-CSF and GM-CSF), non-nucleoside reverse transcriptase inhibitors, nucleoside inhibitors, adriamycin, fluorouracil, methotrexate, asparaginase and combinations thereof. Anti-HIV agents are those in the preceding list that specifically target a function of one or more HIV proteins.
In some embodiments, a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) is administered in combination therapy with two or more anti-HIV agents. For example, a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) can be administered in combination therapy with one, two, or three nucleoside reverse transcriptase inhibitors (e.g., Combivir, Epivir, Hivid, Retrovir, Videx, Zerit. Ziagen, etc.). A SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) can be administered in combination therapy with one or two non-nucleoside reverse transcriptase inhibitors (e.g., Rescriptor, Sustiva, Viramune, etc.). A SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) can be administered in combination therapy with one or two protease inhibitors (e.g., Agenerase, Crixivan, Fortovase, Invirase, Kaletra, Norvir, Viracept, etc.). A SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) can be administered in combination therapy with a protease inhibitor and a nucleoside reverse transcriptase inhibitor. A SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) can be administered in combination therapy with a protease inhibitor, a nucleoside reverse transcriptase inhibitor, and a non-nucleoside reverse transcriptase inhibitor. A SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) can be administered in combination therapy with a protease inhibitor and a non-nucleoside reverse transcriptase inhibitor. Other combinations of a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor) with one or more of a protease inhibitor, a nucleoside reverse transcriptase inhibitor, and a non-nucleoside reverse transcriptase inhibitor are contemplated.
In some embodiments, a treatment method of the present disclosure involves administering: a) a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor); and b) an agent that inhibits an immunodeficiency virus function selected from viral replication, viral protease activity, viral reverse transcriptase activity, viral entry into a cell, viral integrase activity, viral Rev activity, viral Tat activity, viral Nef activity, viral Vpr activity, viral Vpu activity, and viral Vif activity.
In some embodiments, a subject treatment method involves administering: a) a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor); and b) an HIV inhibitor, where suitable HIV inhibitors include, but are not limited to, one or more nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs), fusion inhibitors, integrase inhibitors, chemokine receptor (e.g., CXCR4, CCR5) inhibitors, and hydroxyurea.
Nucleoside reverse transcriptase inhibitors include, but are not limited to, abacavir (ABC; ZIAGEN™), didanosine (dideoxyinosine (ddI); VIDEX™), lamivudine (3TC; EPIVIR™), stavudine (d4T: ZERIT™, ZERIT XR™), zalcitabine (dideoxycytidine (ddC); HMD™), zidovudine (ZDV, formerly known as azidothymidine (AZT): RETROVIR™), abacavir, zidovudine, and lamivudine (TRIZIVIR™), zidovudine and lamivudine (COMBIVIR™), and emtricitabine (EMTRIVA™). Nucleotide reverse transcriptase inhibitors include tenofovir disoproxil fumarate (VIREAD™). Non-nucleoside reverse transcriptase inhibitors for HIV include, but are not limited to, nevirapine (VIRAMUNE™), delavirdine mesylate (RESCRIPTOR™), and efavirenz (SUSTIVA™).
Protease inhibitors (PIs) for treating HIV infection include amprenavir (AGENERASE™), saquinavir mesylate (FORTOVASE™, INVIRASE™), ritonavir (NORVIR™), indinavir sulfate (CRIXIVAN™), nelfmavir mesylate (VIRACEPT™), lopinavir and ritonavir (KALETRA™), atazanavir (REYATAZ™), and fosamprenavir (LEXIVA™).
Fusion inhibitors prevent fusion between the virus and the cell from occurring, and therefore, prevent HIV infection and multiplication. Fusion inhibitors include, but are not limited to, enfuvirtide (FUZEON™), Lalezari et al., New England J. Med., 348:2175-2185 (2003); and maraviroc (SELZENTRY™, Pfizer).
An integrase inhibitor blocks the action of integrase, preventing HIV-1 genetic material from integrating into the host DNA, and thereby stopping viral replication. Integrase inhibitors include, but are not limited to, raltegravir (ISENTRESS™, Merck); and elvitegravir (GS 9137, Gilead Sciences).
Maturation inhibitors include, e.g., bevirimat (3β-(3-carboxy-3-methyl-butanoyloxy) lup-20(29)-en-28-oic acid); and Vivecon (MPC9055).
In some embodiments, a subject treatment method involves administering: a) a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor); and b) one or more of: (1) an HIV protease inhibitor selected from amprenavir, atazanavir, fosamprenavir, indinavir, lopinavir, ritonavir, nelfinavir, saquinavir, tipranavir, brecanavir, darunavir, TMC-126, TMC-114, mozenavir (DMP-450), JE-2147 (AG1776), L-756423, R00334649, KN1-272, DPC-681, DPC-684, GW640385X, DG17, PPL-100, DG35, and AG 1859; (2) an HIV non-nucleoside inhibitor of reverse transcriptase selected from capravirine, emivirine, delaviridine, efavirenz, nevirapine, (+) calanolide A, etravirine, GW5634, DPC-083, DPC-961, DPC-963, MIV-150, and TMC-120, TMC-278 (rilpivirene), efavirenz, BILR 355 BS, VRX 840773. UK-453061, and RDEA806: (3) an HIV nucleoside inhibitor of reverse transcriptase selected from zidovudine, emtricitabine, didanosine, stavudine, zalcitabine, lamivudine, abacavir, amdoxovir, elvucitabine, alovudine, MIV-210, racivir, D-d4FC, emtricitabine, phosphazide, fozivudine tidoxil, apricitibine (AVX754), amdoxovir, KP-1461, and fosalvudine tidoxil (formerly HDP 99.0003): (4) an HIV nucleotide inhibitor of reverse transcriptase selected from tenofovir and adefovir: (5) an HIV integrase inhibitor selected from curcumin, derivatives of curcumin, chicoric acid, derivatives of chicoric acid, 3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, derivatives of aurintricarboxylic acid, caffeic acid phenethyl ester, derivatives of caffeic acid phenethyl ester, tyrphostin, derivatives of tyrphostin, quercetin, derivatives of quercetin, 5-1360, zintevir (AR-177), L-870812, and L-870810, MK-0518 (raltegravir), BMS-538158, GSK364735C, BMS-707035, MK-2048, and BA 011; (6) a gp41 inhibitor selected from enfuvirtide, sifuvirtide, FB006M, and TRI-1144; (7) a CXCR4 inhibitor, such as AMD-070; (8) an entry inhibitor, such as SPOIA; (9) a gp120 inhibitor, such as BMS-488043 and/or BlockAide/CR: (10) a G6PD and NADH-oxidase inhibitor, such as immunitin; (11) a CCR5 inhibitors selected from the group consisting of aplaviroc, vicriviroc, maraviroc. PRO-140, INCB15050, PF-232798 (Pfizer), and CCR5 mAbtX4; (12) another drug for treating HIV selected from BAS-100, SPI-452, REP 9, SP-01A, TNX-355, DES6, ODN-93, ODN-112, VGV-1, PA-457 (bevirimat), Ampligen, HRG214, Cytolin, VGX-410, KD-247, AMZ 0026, CYT 99007A-221 HIV, DEBIO-025, BAY 50-4798, MDXO10 (ipilimumab), PBS119, ALG 889, and PA-1050040 (PA-040); (13) any combinations or mixtures of the above.
As further examples, in some embodiments, a subject treatment method involves administering: a) a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor); and b) one or more of: i) amprenavir (Agenerase; (3S)-oxolan-3-yl N-[(2S,3R)-3-hydroxy-4-[N-(2-methylpropyl)(4-aminobenzene)sulfonamido]-1-phenylbutan-2-yl]carbamate) in an amount of 600 mg or 1200 mg twice daily: ii) tipranavir (Aptivus; N-{3-[(1R)-1-[(2R)-6-hydroxy-4-oxo-2-(2-phenylethyl)-2-propyl-3,4-dihydro-2H-pyran-5-yl]propyl]phenyl}-5-(trifluoromethyl)pyridine-2-sulfonamide) in an amount of 500 mg twice daily; iii) idinavir (Crixivan: (2S)-1-[(2S,4R)-4-benzyl-2-hydroxy-4-{[(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]carbamoyl}butyl]-N-tert-butyl-4-(pyridin-3-ylmethyl)piperazine-2-carboxamide) in an amount of 800 mg three times daily; iv) saquinavir (Invirase: 2S)—N-[(2S,3R)-4-[(3S)-3-(tert-butylcarbamoyl)-decahydroisoquinolin-2-yl]-3-hydroxy-1-phenylbutan-2-yl]-2-(quinolin-2-ylformamido)butanediamide) in an amount of 1,000 mg twice daily; v) lopinavir and ritonavir (Kaleta: where lopinavir is 2S)—N-[(2S,4S,5S)-5-[2-(2,6-dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-(2-oxo-1,3-diazinan-1-yl)butanamide; and ritonavir is 1,3-thiazol-5-ylmethyl N-[(2S,3S,5S)-3-hydroxy-5-[(2S)-3-methyl-2-{[methyl({[2-(propan-2-yl)-1,3-thiazol-4-yl]methyl})carbamoyl]amino}butanamido]-1,6-diphenylhexan-2-yl]carbamate) in an amount of 133 mg twice daily; vi) fosamprenavir (Lexiva; {[(2R,3S)-1-[N-(2-methylpropyl)(4-aminobenzene)sulfonamido]-3-({[(3S)-oxolan-3-yloxy]carbonyl}amino)-4-phenylbutan-2-yl]oxy}phosphonic acid) in an amount of 700 mg or 1400 mg twice daily): vii) ritonavir (Norvir) in an amount of 600 mg twice daily; viii) nelfinavir (Viracept; (3S,4aS,8aS)—N-tert-butyl-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylphenyl)formamido]-4-(phenylsulfanyl)butyl]-decahydroisoquinoline-3-carboxamide) in an amount of 750 mg three times daily or in an amount of 1250 mg twice daily; ix) Fuzeon (Acetyl-YTSLIHSLIEESQNQ QEKNEQELLELDKWASLWNWF-amide (SEQ ID NO:21)) in an amount of 90 mg twice daily; x) Combivir in an amount of 150 mg lamivudine (3TC; 2′,3′-dideoxy-3′-thiacytidine) and 300 mg zidovudine (AZT; azidothymidine) twice daily: xi) emtricitabine (Emtriva; 4-amino-5-fluoro-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one) in an amount of 200 mg once daily; xii) Epzicom in an amount of 600 mg abacavir (ABV; {(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]cyclopent-2-en-1-yl}methanol) and 300 mg 3TC once daily; xiii) zidovudine (Retrovir; AZT or azidothymidine) in an amount of 200 mg three times daily: xiv) Trizivir in an amount of 150 mg 3TC and 300 mg ABV and 300 mg AZT twice daily; xv) Truvada in an amount of 200 mg emtricitabine and 300 mg tenofovir (({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid) once daily: xvi) didanosine (Videx; 2′,3′-dideoxyinosine) in an amount of 400 mg once daily; xvii) tenofovir (Viread) in an amount of 300 mg once daily: xviii) abacavir (Ziagen) in an amount of 300 mg twice daily; xix) atazanavir (Reyataz; methyl N-[(1S)-1-{[(2S,3S)-3-hydroxy-4-[(2S)-2-[(methoxycarbonyl)amino]-3,3-dimethyl-N′-{[4-(pyridin-2-yl)phenyl]methyl}butanehydrazido]-1-phenylbutan-2-yl]carbamoyl}-2,2-dimethylpropyl]carbamate) in an amount of 300 mg once daily or 400 mg once daily; xx) lamivudine (Epivir) in an amount of 150 mg twice daily; xxi) stavudine (Zerit: 2′-3′-didehydro-2′-3′-dideoxythymidine) in an amount of 40 mg twice daily; xxii) delavirdine (Rescriptor; N-[2-({4-[3-(propan-2-ylamino)pyridin-2-yl]piperazin-1-yl}carbonyl)-1H-indol-5-yl]methanesulfonamide) in an amount of 400 mg three times daily; xxiii) efavirenz (Sustiva; (4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-2,4-dihydro-1H-3,1-benzoxazin-2-one) in an amount of 600 mg once daily); xxiv) nevirapine (Viramune; I l-cyclopropyl-4-methyl-5,11-dihydro-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one) in an amount of 200 mg twice daily); xxv) bevirimat; and xxvi) Vivecon.
In some embodiments, a subject treatment method involves administering: a) a SMYD2 inhibitor (and/or an ASH1L inhibitor, and/or an SUV420H1 inhibitor, and/or an SUV39H1 inhibitor); and b) a PKC activator. An example of a suitable PKC activator is prostratin ((1aR,1bS,4aR,7aS,7bR,8R,9aS)-4a,7b-dihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-5-oxo-1,1a,1b,4,4a,5,7a,7b,8,9-decahydro-9aH-cyclopropa[3,4]benzo[1,2-e]azulen-9a-yl). The PKC activator can be administered in a separate formulation from a SMYD2 inhibitor. A PKC activator can be co-formulated with a SMYD2 inhibitor, and the co-formulation administered to an individual. The present disclosure provides a kit comprising a PKC activator in a first container; and a SMYD2 inhibitor in a second container.
Subjects Suitable for Treatment
The methods of the present disclosure are suitable for treating individuals who have an immunodeficiency virus infection, e.g., who have been diagnosed as having an immunodeficiency virus infection.
The methods of the present disclosure are suitable for treating individuals who have an HIV infection (e.g., who have been diagnosed as having an HIV infection), and individuals who are at risk of contracting an HIV infection. Such individuals include, but are not limited to, individuals with healthy, intact immune systems, but who are at risk for becoming HIV infected (“at-risk” individuals). At-risk individuals include, but are not limited to, individuals who have a greater likelihood than the general population of becoming HIV infected. Individuals at risk for becoming HIV infected include, but are not limited to, individuals at risk for HIV infection due to sexual activity with HIV-infected individuals. Individuals suitable for treatment include individuals infected with, or at risk of becoming infected with, HIV-1 and/or HIV-2 and/or HIV-3, or any variant thereof.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s): kb, kilobase(s): pl, picoliter(s); s or sec, second(s); min. minute(s); h or hr, hour(s), aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly): i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
Materials and Methods
HEK293T and Jurkat cells were obtained from the American Type Culture Collection. J-Lat (clones A2 and A72) cell lines were cultured as described in Jordan el al., EMBO J. 2003 Apr. 15:22(8):1868-77. HEK293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin (Life Technologies). Tumor necrosis factor-alpha (TNFα) (Sigma-Aldrich) was used at concentrations of 0.5 or 1 ng/ml. Human αCD3/αCD28 beads (Life Technologies) were used at a concentration of 1 bead/cell ratio.
ShRNA-Mediated Knockdown Experiments and Flow Cytometry Analysis
ShRNA-expressing lentiviral vectors were purchased from Open Biosystems. The plasmids TRCN0000276155, TRCN0000276082, TRCN0000276083, TRCN0000276085, TRCN0000130774, TRCN0000130403, and TRCN0000128349 were used to deplete SMYD2. The pLKO.1 vector containing scramble shRNA was used as control. Pseudotyped viral stocks were produced in 2×106 HEK293T cells by the calcium phosphate method by co-transfection of 10 μg of shRNA-expressing lentiviral vectors, together with 6.5 μg of the lentiviral packaging construct pCMVdelta R8.91 and 3.5 μg of VSV-G glycoprotein-expressing vector, and titered for p24 content. J-Lat A72 cells (containing a long terminal repeat (LTR)-green fluorescent protein (GFP) (LTR-GFP) construct) were spin-infected with virus (1 ng of p24 per 106 cells) containing shRNAs against SMYD2 or nontargeting control shRNAs; infected cells were selected with puromycin (2 μg/ml: Sigma). After 4 days of selection, cells were treated with the indicated concentration of drugs. The percentage of GFP+ cells was determined after 18 h using a MACSQuant VYB fluorescence activated cell sorting (FACS) analyzer (Miltenyi Biotech GmbH). Cell viability was monitored by forward and side scatter analysis. Analysis was conducted on 3×20,000 live cells per condition, and all experiments were independently repeated at least three times. Data were analyzed using FlowJo 9.4 (Tree Star). Nucleotide sequences of SMYD2 shRNAs, scramble control shRNA, and luciferase control shRNA are provided in
In Vitro Methylation Assays
For protein reactions, 2 μg of histones (isolated from HEK293T cells), and synthetic Tat protein were incubated overnight at 30° C. with recombinant SMYD2 (Sigma) in a buffer containing 50 mM TrisHCl pH 9, 0.01% Tween 20, 2 mM dithiothreitol (DTT) and 1.1 μCi of 3H-labeled SAM (Perkin Elmer). Peptide reactions contained 2 μg of each peptide and recombinant SMYD2. Reaction mixtures were fractionated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for proteins or on 10-20% Tris-Tricine gradient gels for peptides (BioRad). After coomassie staining, gels were treated with Amplify (GE Healthcare) for 30 min, dried and exposed to hyperfilm (GE Healthcare) overnight.
Use of Polyclonal Anti-meARM Antibodies
The anti-meARM (α-meARM) antibodies were generated in rabbits immunized with chemically synthesized K51-monomethylated ARM. For western blotting of synthetic Tat proteins, biotinylated synthetic Tat was incubated in the presence or absence of SMYD2 enzyme and nonradioactive SAM.
Primary T-Cell Model of HIV Latency (“Greene Model”)
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation of buffy coats from HIV-seronegative donors (Blood Centers of the Pacific). PBMCs were immediately processed to isolate CD4+ T cells. Total CD4+ T cells were isolated by negative selection, according to manufacturer's protocol, with the EasySep CD4+ T-cell Enrichment Kit (Stem Cell Technologies). Isolated CD4+ T cells were cultured in RPMI as described above at a concentration of 1×106 cells/ml for 24 h before HIV infection.
CD4+ T cells were counted, collected as pellets by centrifugation at 1500 rpm for 5 min at room temperature, and resuspended in the appropriate volume of concentrated viral NL4-3-Luc supernatant. Typically, 50-200 ng of p24Gag per 4×105 CD4+ T cells were used. Spinoculations with NL4-3-Luc virus were performed in 96-well V-bottom plates with up to 5×105 CD4+ T cells per well. All spinoculations were performed in volumes of 200 μl or less. Cells and virus were centrifuged at 2000 rpm for 1.5-2 h at room temperature. After spinoculation, cells were pooled and cultured at a concentration of 1×106 cells/ml in RPMI 1640 containing 10% FBS and supplemented with 5 μM saquinavir for 3 days to prevent residual spreading infection. Saquinavir was purchased from Sigma.
For reactivation of latent HIV-1 provirus, cells were counted and collected as pellets by centrifugation at 1500 rpm for 10 min. Cells were then plated in 96-well U-bottom plates at concentrations of 1×106/200 μl in the presence of the indicated activator. Unless otherwise indicated, cells were cultured either in medium alone or stimulated with, 5 μg/ml phytohemagglutinin (PHA) (Sigma), 10 ng/ml TNF-α, or anti-CD3+anti-CD28 beads at a ratio of 1:1. Cells were harvested 48 hr after stimulation, washed one time with phosphate buffered saline (PBS), and lysed in 60 μl of Cell Lysis Buffer (Promega) After 15 min of lysis, the luciferase activity in cell extracts was quantified with a BD Monolight Luminometer after mixing 20 μl of lysate with 100 μl of substrate (Luciferase Assay System-Promega). Relative light units were normalized to protein content determined by BCA assay (Pierce). Cell survival rates were measured by flow cytometry immediately before lysis.
Results
Knockdown of SMYD2 Reactivates HIV-LTR
To test the functional relevance of SMYD2 in HIV latency, lentiviral shRNA knockdown studies of endogenous SMYD2 proteins were performed in a J-Lat cell line harboring a latent lentiviral construct expressing Tat with GFP from the HIV LTR (clone A2; LTR-Tat-IRES-GFP). As shown in
SMYD2 Methylates Tat at K51
As SMYD2 is known as a protein methyltransferase (p53, Rb), it was tested whether Tat is methylated by SMYD2. Full-length synthetic Tat protein (aa 1-72) was incubated with recombinant SMYD2 enzyme and radiolabeled S-adenosyl-L-methionine (SAM). Reactions were resolved by gel electrophoresis and developed by autoradiography. As shown in
To map the site of methylation in Tat, short synthetic Tat peptides were subjected to in vitro methylation assays. As shown in
Materials and Methods
J-Lat (clones A2 and A72) cell lines were cultured as described in Jordan et al., EMBO J. 2003 Apr. 15:22(8):1868-77. Human αCD3/αCD28 beads (Life Technologies) were used at a concentration of 1 bead/cell ratio. JQ1 (Sigma-Aldrich) was used at a concentration of 0.1-10 μM. Ingenol 3,20-dibenzoate (Sigma-Aldrich) was used at concentrations of 5-200 nM, and SAHA (Sigma-Aldrich) was used at concentrations of 110 nM, 330 nM, or 1 μM.
Primary T-Cell Model of HIV Latency (“Greene Model”)
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation of buffy coats from HIV-seronegative donors (Blood Centers of the Pacific). PBMCs were immediately processed to isolate CD4+ T cells. Total CD4+ T cells were isolated by negative selection, according to manufacturer's protocol, with the EasySep CD4+ T-cell Enrichment Kit (Stem Cell Technologies). Isolated CD4+ T cells were cultured in RPMI as described above at a concentration of 1×106 cells/ml for 24 h before HIV infection.
CD4+ T cells were counted, collected as pellets by centrifugation at 1500 rpm for 5 min at room temperature, and resuspended in the appropriate volume of concentrated viral NL4-3-Luc supernatant. Typically, 50-200 ng of p24Gag per 4×105 CD4+ T cells were used. Spinoculations with NL4-3-Luc virus were performed in 96-well V-bottom plates with up to 5; 10 HLAC or CD4 T cells per well. All spinoculations were performed in volumes of 200 μl or less. Cells and virus were centrifuged at 2000 rpm for 1.5-2 h at room temperature. After spinoculation, cells were pooled and cultured at a concentration of 1×106 cells/ml in RPMI 1640 containing 10% FBS and supplemented with 5 μM saquinavir for 3 days to prevent residual spreading infection. Saquinavir was purchased from Sigma.
For reactivation of latent HIV-1 provirus, cells were counted and collected as pellets by centrifugation at 1500 rpm for 10 min. Cells were then plated in 96-well U-bottom plates at concentrations of 1×106/200 μl in the presence of the indicated activator. Unless otherwise indicated, cells were cultured either in medium alone or stimulated with 5 μg/ml phytohemagglutinin (PHA) (Sigma), 10 ng/ml TNF-α, anti-CD3+anti-CD28 beads at a ratio of 1:1. SAHA, JQ1 and X2 were tested at the indicated concentrations. Cells were harvested 48 hr after stimulation, washed one time with PBS, and lysed in 60 μl of Cell Lysis Buffer (Promega). After 15 min of lysis, the luciferase activity in cell extracts was quantified with a BD Monolight Luminometer after mixing 20 μl of lysate with 100 μl of substrate (Luciferase Assay System-Promega). Relative light units were normalized to protein content determined by BCA assay (Pierce). Cell survival rates were measured by flow cytometry immediately before lysis.
Results
Small-Molecule SMYD2 Inhibitors Reactivate HIV in J-Lat Cell Lines
As SMYD2 knockdown shows reactivation potential at the HIV LTR, it was speculated that treatment with SMYD2 inhibitors might activate Tat transcriptional activity and reactivate HIV from latency. To test this hypothesis, J-Lat cells (clone A2: LTR-Tat-IRES-GFP) were treated with SMYD2 inhibitors. As shown in
SMYD2 Inhibitor X2 Co-Treatment Reactivates HIV in a Primary CD4+ T Cell Model
Since X2 activated HIV from latency in A2 and A72 cell lines, this compound was tested in a primary T-cell model of latency (Lassen, Greene). In this model, CD4+ T cells were infected in a single-round infection with HIV clone NL4-3-Luc to generate a latent infection in vitro. To reactivate latent HIV-1, cells were treated with the indicated compounds or a combination of PHA/IL-2 as a control for maximal activation. X2 in combination with JQ1 reactivated latent HIV-1 at 8-25% of the rate achieved by costimulation with PHA/IL-2 (
Materials and Methods
HEK293T cells were obtained from the American Type Culture Collection. J-Lat (clones A2, A72, and 5A8) cell lines were described (Chan et al., 2013; Jordan et al., 2003). HEK293T cells were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin (Life Technologies). J-Lat cells were cultured in RPMI supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin (Life Technologies). SMYD2 and RELA antibodies were purchased from Bethyl, Histone 4, H4K20me1, H4K20me2, and H4K20me3 antibodies were purchased from Active Motif, and rabbit IgG isotype control (10500C) was purchased from Thermo Fisher Scientific. TNFα (Sigma-Aldrich) was used at 0.5-10 ng/ml. Human αCD3/αCD28 Dynabeads (Invitrogen) were used at a 1 bead/cell ratio. JQ1 (Cayman Chemical) was used at 0.1-10 μM. Ingenol 3,20-dibenzoate (Santa Cruz Biotechnology) was used at 5-200 nM, and SAHA (Merck) was used at 110 nM, 330 nM, or 1 μM. Phorbol 12-myristate 13-acetate (PMA) (Sigma) was used at 10 nM and ionomycin (Sigma) was used at a concentration of 500 nM. UNC926 (Tocris Bioscience) was used at a concentration of 10 nM-100 μM. AZ505. AZ506, and AZ391 were used at a concentration of 10 nM-10 μM.
ShRNA-Mediated Knockdown Experiments and Flow Cytometry Analysis
ShRNA-expressing lentiviral vectors were purchased from Sigma-Aldrich. The plasmids used in the shRNA screen are listed in Table 1 below. The pLKO.1 vector containing a scrambled shRNA was used as control. Pseudotyped viral stocks were produced in 2×106 HEK293T cells by the calcium phosphate method by co-transfecting 10 μg of shRNA-expressing lentiviral vectors, with 6.5 μg of the lentiviral packaging construct pCMVdelta R8.91 and 3.5 μg of VSV-G glycoprotein-expressing vector (Naldini et al., 1996), and titered for p24 content. J-Lat 5A8, A72 and A2 cells were spininfected with virus (1 ng of p24 per 106 cells) containing shRNAs against KMTs or nontargeting control shRNAs and were selected with puronycin (2 μg/ml: Sigma). After 7 days of selection, cells were treated with the indicated concentration of drugs. The percentage of GFP+ cells was determined after 18 h using a MACSQuant VYB FACS analyzer (Miltenyi Biotech GmbH). Cell viability was monitored by forward-and-side scatter analysis. Analysis was conducted on 3×10,000 live cells per condition. Data were analyzed using FlowJo 9.5 (Tree Star).
In Vitro Methylation Assays
In vitro Methylation assays were performed as described (Nishioka et al., 2002). For reactions, 2 gtg of histones (isolated from HEK293T cells), recombinant histone 4 (New England Biolabs), synthetic histone 4 as 1-21 and as 15-24 peptides (Cayman Chemical), or synthetic histone H4 as 1-21 with a K20A mutation (GenScript) were incubated with recombinant WT SMYD2 (Sigma-Aldrich) or SMYD2 Y240F (Active Motif) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3-labeled SAM (Perk-in Elmer) overnight at 30° C. Reaction mixtures were fractionated on 15% SDS-PAGE for proteins or on 10-20% Tris-Tricine gradient gels for peptides (BioRad). After Coomassie staining, gels were treated with Amplify. (GE Healthcare) for 30 min, dried and exposed to hyperfilm (GE Healthcare) overnight.
Experiments with Primary CD4+ Cells from Latently HIV-1-Infected Individuals
Four aviremic HIV-1-infected individuals were recruited from the SCOPE cohorts at the University of California. San Francisco. Table 2 details the characteristics of the study participants.
Peripheral blood mononuclear cells (PBMCs) from whole blood or continuous flow centrifugation leukapheresis product were purified using density centrifugation on a Ficoll-Hypaque gradient. Resting CD4+ lymphocytes were enriched by negative depletion with an EasySepHuman CD4+ T Cell Isolation Kit (Stemcell). Cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and 5 μM saquinavir. Five million resting CD4′ lymphocytes were stimulated with latency-reversing agents (LRAs) at the indicated concentrations (20-500 nM AZ391, 100 nM JQ1, 25 μl/1×106 T cells αCD3/αCD28 Dynabeads (Life Technologies) for 48 hours. After LRA treatment, cells were collected, lysed and total RNA was isolated with an RNeasy kit (Quiagen). A Superscript III One-Step RT-PCR system (Life Technologies) was used to generate and pre-amplify cell-associated viral mRNA. Reaction mixes contained 15 μl of a PCR mix containing reaction mix, Superscript III, primers (900 nM final concentration) and 10 μl purified RNA. Pre-amplification was carried out using the following steps: reverse transcription at 50° C. for 30 min, denaturation at 95° C. for 2 min, 10 cycles of amplification (94° C. 15 s, 55° C. 30 s, 68° C. 5 min) on a GeneAmp PCR system 9700 (Thermo Fisher). Subsequently, droplet digital PCR (ddPCR) was applied to quantify pre-amplified cDNA. Each 25 μl ddPCR mix comprised the ddPCR Probe Supermix (no dUTP), 900 nM primers, 250 nM probe, and 4 μl cDNA. The following cycling conditions were used: 10 minutes at 95° C. 40 cycles each consisting of 30 second denaturation at 94° C. followed by 59.4° C. extension for 60 seconds, and a final 10 minutes at 98° C. Reaction mixes were loaded into the Bio-Rad QX-100 emulsification device and droplets were formed following the manufacturer's instructions. Then, samples were transferred to a 96-well reaction plate and sealed with a pre-heated Eppendorf 96-well heat sealer for 2 seconds, as recommended by Bio-Rad. Finally, samples were amplified on a BioRad C1000 Thermocycler and analyzed using a BioRad QX100 ddPCR Reader.
Nucleotide coordinates are indicated relative to HXB2 consensus sequence. Primers and probe used for HIV-1 mRNA measurement were as described (Laird et al., 2015):
RNA Isolation, Reverse Transcription, and Quantitative RT-PCR
RNA was isolated using RNeasy Plus Mini Kit (Qiagen) and reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) as per the manufacturer's instructions. Quantitative RT-PCR was carried out using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) on SDS 2.4 software (Applied Biosystems) in a total volume of 12 μL. Primer efficiencies were around 100%. Dissociation curve analysis was performed after the end of the PCR to confirm the presence of a single and specific product.
Chromatin Immunoprecipitation
J-Lat A2 and A72 cells were treated with TNFα (10 ng/ml) for 18 h. Cells were fixed with 1% formaldehyde (v/v) in fixation buffer (1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes, pH 8.0, 100 mM NaCl), and fixation was stopped after 10 min by addition of glycine to 125 mM. The cell membrane was lysed for 15 min on ice (5 mM Pipes, pH 8.0, 85 mM KCl, 0.5% NP40, protease inhibitors). After washing with nuclear swell buffer (25 mM HEPES, pH 7.5, 4 mM KCl, 1 mM DTT, 0.5% NP-40, 0.5 mM PMSF) and micrococcal nuclease (MNase) digestion buffer (20 mM Tris pH 7.5, 2.5 mM CaCl2), 5 mM NaCl, 1 mM DTT, 0.5% NP-40), the pellet was resuspended in MNase buffer (15 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM CaCl2, and 25 mM NaCl). Subsequently, samples were incubated with MNase (New England Biolabs) for 10 mm at RT. The reaction was quenched with 0.5 M EDTA and incubated on ice for 5 min. Cells were lysed (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, protease inhibitors), and chromatin DNA was sheared to 200-1000-bp average size through sonication (Ultrasonic Processor CP-130, Cole Parmer). Cellular debris was pelleted, and the supernatant was recovered. Protein A/G Sepharose beads were blocked with single-stranded salmon sperm DNA and BSA, washed and resuspended in immunoprecipitation buffer. Blocked protein A/G Sepharose beads were added to the digested chromatin fractions and rotated at 4° C. for 2 h to preclear chromatin. Lysates were incubated overnight at 4° C. with 5 μg of SMYD2, RELA, histone H4, H4K20me1, H4K20me2, H4K20me3 antibodies, or IgG control. After incubation with protein A/G agarose beads for 2 h and washing three times with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), one time with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl) and twice with TE-buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.1), chromatin was eluted and recovered with Agencourt AMPure XP beads (Beckman Coulter). Bound chromatin and input DNA were treated with RNase H (New England Biolabs) and Proteinase K (Sigma-Aldrich) at 37° C. for 30 min. Immunoprecipitated chromatin was quantified by real-time PCR using the Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and the ABI 7700 Sequence Detection System (Applied Biosystems). The SDS 2.4 software (Applied Biosystems) was used for analysis. The specificity of each PCR reaction was confirmed by melting curve analysis using the Dissociation Curve software (Applied Biosystems). All chromatin immunoprecipitations and qPCRs were repeated at least three times, and representative results were shown.
Primer sequences were:
Mass Spectrometry Analysis
Samples were denatured and reduced in 2 M urea, 10 mM NH4HCO3, 2 mM DTT for 30 min at 60° C., then alkylated with 2 mM iodoacetamide for 45 min at room temperature. Samples were then digested with 0.5 μg of LysC (Roche) overnight at 37 C. Following digestion, samples were concentrated using C18 ZipTips (Millipore) according to the manufacturer's specifications. Desalted samples were evaporated to dryness and resuspended in 0.1% formic acid for mass spectrometry analysis.
Digested samples were analyzed in technical duplicate on a Thermo Fisher Orbitrap Fusion mass spectrometry system equipped with a Easy nLC 1200 ultra-high pressure liquid chromatography system interfaced via a Nanospray Flex nanoelectrospray source. Samples were injected on a C18 reverse phase column (25 cm×75 um packed with ReprosilPur C18 AQ 1.9 um particles). Peptides were separated by an organic gradient from 5-30% ACN in 0.1% formic acid over 112 minutes at a flow rate of 300 nl/min. The MS continuously acquired spectra in a data-dependent manner throughout the gradient, acquiring a full scan in the Orbitrap (at 120,000 resolution with an AGC target of 200,000 and a maximum injection time of 100 ms) followed by as many MS/MS scans as could be acquired on the most abundant ions in 3 s in the dual linear ion trap (rapid scan type with an intensity threshold of 5000, HCD collision energy of 29%, AGC target of 10,000, a maximum injection time of 35 ms, and an isolation width of 1.6 m/z). Singly and unassigned charge states were rejected. Dynamic exclusion was enabled with a repeat count of 1, an exclusion duration of 20 s, and an exclusion mass width of +/−10 ppm.
Raw mass spectrometry data were assigned to histone H4 sequences with the MaxQuant software package (version 1.5.5.1) (Cox and Mann, 2008). Variable modifications were allowed for N-terminal protein acetylation, methionine oxidation, and lysine methylation. A static modification was indicated for carbamidomethyl cysteine. All other settings were left as MaxQuant defaults. MaxQuant-identified peptides were quantified by MS1 filtering using the Skyline software suite (MacLean et al., 2010).
Ex Vivo Infection of Tonsil-Derived (HLAC) Cells
HLAC cells were isolated by Ficoll-Histopaque density gradient centrifugation of sheared tonsils from HIV-seronegative donors (Vanderbilt University Medical Center, Nashville, Tenn.). Isolated HLAC cells were counted, collected as pellets by centrifugation at 1500 rpm for 5 min at room temperature, and re-suspended in the appropriate volume of concentrated viral NL4.3-Luc supernatant. Typically, 50-100 ng of p24 Gag per 4×105 HLAC were used. Spinoculations were performed in 96-well V-bottom plates in volumes of 200 μl or less. Cells and virus were centrifuged at 2000 rpm for 1.5-2 h at room temperature. After spinoculation, cells were pooled and cultured at 1×106 cells/ml in RPMI 1640 containing 10% FBS and supplemented with 5 μM Saquinavir (Sigma-Aldrich) for 3 days to prevent any residual spreading infection.
For reactivation of latent HIV-1 provirus, cells were counted and collected as pellets by centrifugation at 1500 rpm for 10 min. Cells were then plated in 96-well U-bottom plates at 1×106 per 200 μl in the presence of 30 μM Raltegravir (Santa Cruz Biotechnology) and the indicated activator. Cells were harvested 48 h after stimulation, washed one time with PBS, and lysed in 60 μl of Passive Lysis Buffer (Promega). After 15 min of lysis, the luciferase activity in cell extracts was quantified with a Perkin Elmer EnSpire 2300 Multimode plate reader after mixing 20 μl of lysate with 100 μl of substrate (Luciferase Assay System-Promega). Relative light units (RLU) were normalized to protein content determined by Bradford assay (BioRad). Cell viability was measured with CellTiter-Blue Cell Viability Assay (Promega).
T-Cell Activation Analysis
Human CD4+ T cells isolated from blood (Blood Centers of the Pacific, San Francisco, Calif.) by negative selection using RosetteSep Human CD4+ T Cell Enrichment Cocktail (StemCell Technologies) were incubated for 24 h in 6-well plates with AZ391 (1 μM), JQ1 (500 nM), or IL-2 (20 U/ml), all dissolved in DMSO at a 1:10,000 dilution. CD69 and CD25 expression was measured by flow cytometry gating on CD3+CD4+ T cells using FITC-labeled antibodies for CD3 (11-0048-42, eBioscience), APC-conjugated CD25 antibodies (17-0259-42, eBioscience), PerCP-labeled antibodies for CD4 (300528, Biolegend), and CD69-V450 (560740, BD Horizon). Staining was performed for 30 min on ice in FACS buffer (PBS, 2% FBS), and samples were analyzed on a BD Biosciences LSRII flow cytometer.
Results
ShRNA Screen Identifies Novel KMTs Involved in HIV-1 Latency
To identify novel epigenetic regulators of HIV latency, small hairpin RNAs (shRNAs) that target 31 cellular KMTs were screened in the CD4+ J-Lat 5A8 cell line harboring a latent full-length HIV provirus with the fluorescent marker GFP inserted into the nef open-reading frame to allow monitoring of transcriptional activity by flow cytometry (
Four KMTs were identified as repressors of HIV latency, as their knockdown with both shRNAs induced transcriptional activation (ASH1L. SMYD2, SUV39H1, and SUV420H1). EZH1, a component of the PRC2 complex linked to HIV latency (Friedman et al., 2011), showed hyperactivation only after high-dose αCD3/28 treatment. Twelve KMTs were identified as coactivators of the reactivation response, including SET7/9, which was previously identified as a transcriptional activator of HIV that methylates the viral transactivator Tat (Pagans et al., 2010). To independently confirm repressive activities of ASH1L, SMYD2, SUV39H1, and SUV420H1, the screen was repeated in two other J-Lat clones, A72 and A2. These clones contain HIV minigenomes composed of just the HIV promoter in the 5′LTR that drives GFP expression (LTR-GFP; A72) or an LTR-Tat-IRES-GFP cassette where transcriptional activity is driven by the viral transactivator Tat (A2) (Jordan et al., 2003; Jordan et al., 2001). In both cells lines, spontaneous latency reversal (≥2× increase in GFP+ cells) was observed in cells lacking SMYD2, ASH1L, SUV420H1, and SUV39H1, with SMYD2 representing the top hit in both cell lines (
Inhibition of SMYD2 Reactivates HIV-1 from Latency
Because of SMYD2's role in p53 and RB tumor suppressor inactivation and cancer development (Hamamoto et al., 2015; Huang et al., 2006), a specific SMYD2 inhibitor (AZ505) was developed (Ferguson et al., 2011). AZ505 is a substrate-competitive inhibitor that binds the peptide-binding groove of the enzyme with a calculated Kd of 0.5 μM, approximately sevenfold lower than the p53 peptide. AZ505 is not cell-penetrable, but subsequent efforts identified a novel series of potent, cell-permeable SMYD2 inhibitors, including analogs AZ506 (IC50=0.017 μM) and AZ391 (IC50=0.027 μM) (Cowen, 2013; Throner, 2015). The ability of these compounds to reverse HIV latency was tested in the J-Lat A72 cell line. Indeed, both compounds, but not AZ505, activated GFP expression at high concentrations (5 and 10 μM), with AZ391 inducing up to 30% GFP+ cells similar to the activity of TNFα or the BET inhibitor JQ1 (
Next, AZ391 was tested in CD4+ T cells from HIV-1-infected individuals on suppressive ART. Four HIV-1-infected individuals, who met the criteria of suppressive ART, which is undetectable plasma HIV-1 RNA levels (<50 copies/ml) for a minimum of six months, and a CD4+ T cell count of at least 350 cells/mm3, were enrolled (Table 2). In a pilot experiment, five million purified CD4+ T cells from one individual were treated ex vivo with increasing, non-toxic concentrations of AZ391 (maximal 500 nM), JQ1 or a combination of both, or vehicle alone. After 48 hours, levels of intracellular HIV-1 mRNA were measured by droplet digital RT-PCR using a previously published primer/probe set (Laird et al., 2015). AZ391 treatment increased intracellular HIV-1 mRNA levels in a dose-dependent manner to a similar extent as JQ1: however, no additive or synergistic effects between both drugs were observed (
SMYD2 Associates with the HIV Promoter in Cells
To examine SMYD2's association with the latent HIV promoter, ChIP experiments were used. Chromatin was prepared from J-Lat A72 cells, either unstimulated or stimulated with TNFα, incubated with a ChIP-grade SMYD2 or IgG control antibodies, and immunoprecipitated as described (Schroder et al., 2013). DNA extracted from the immunoprecipitated material or the input control, and quantitative PCR analysis was performed with primers specific for the region within the HIV promoter occupied by nuc-1 or for the irrelevant AXIN2 gene (Kaehlcke et al., 2003). Significant enrichment over the input and the IgG control was observed for SMYD2 at the HIV LTR, but not at the AXIN2 gene, demonstrating specific association of SMYD2 with the latent promoter (
SMYD2 Monomethylates Lysine 20 in Histone 4
To identify the target for SMYD2 at the latent HIV promoter, in vitro methylation assays were performed with recombinant SMYD2 and radio-labeled S-adenosyl methionine (SAM) on purified human histones. Histone H4 was prominently methylated by SMYD2 (
To determine if K20 is the site of methylation in H4, in vitro methylation assays were performed with a K20A-mutated histone H4 peptide. K20 was efficiently methylated by SMYD2 in the wildtype peptide, a process abolished by the H4K20A mutation (
As antibodies against the different methylated states of H4K20 are readily available, ChIP analysis was next performed in A72 cells followed by qPCR specific for the HIV promoter. It was found that, like SMYD2, H4K20me1, but not H4K20me2/3, was markedly enriched at the latent HIV promoter (
Next, ChIP analysis was performed in SMYD2 knockdown A72 cells. SMYD2 knockdown and confirmed by western blotting (
Recruitment of Reader Protein L3MBTL1 to the Latent HIV-1 Promoter
L3MBTL1 is an MBT (malignant brain tumor) family member, a highly conserved group of 11 proteins characterized by multiple MBT domains that together bind mono- and dimethylated histones (Bonasio et al., 2010). H4K20me1/2 was identified as a docking site for L3MBTL1 in chromatin by the Reinberg laboratory, who also documented chromatin-compacting properties for purified L3MBTL1 on reconstituted nucleosomal arrays (Trojer et al., 2007). To determine if the chromatin-compacting activity of L3MBTL1 is recruited to the latent HIV promoter, ChIP experiments were performed with L3MBTL1 antibodies and found L3MBTL1 enriched at latent and disenriched at the TNFα-activated HIV promoter in A72 (
Materials and Methods
HEK293T cells were obtained from the American Type Culture Collection. J-Lat (clones A2, A72, and 5A8) have been previously described. HEK293T cells were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin (Life Technologies). J-Lat cells were cultured in RPMI supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin (Life Technologies). Histones were isolated from HEK293T cells. The recombinant Tat peptides were synthesized by PSL Peptide Specialty Laboratories GmbH (German Cancer Research Center). Recombinant p65, SPI, CyclinT1/CDK9 and SMYD5 were purchased from Active Motif. Human αCD3/αCD28 Dynabeads (Invitrogen) were used at a 1 bead/cell ratio.
ShRNA-Mediated Knockdown Experiments, Flow Cytometry Analysis
ShRNA-expressing lentiviral vectors were purchased from Sigma-Aldrich. The plasmids TRCN000155095 (Target sequence: GCTATGGGAATTACAACCCAT) (SEQ ID NO:76) and TRCN0000156306 (Target sequence: CTGTGACACTCTGGAGTTGAA) (SEQ ID NO:77) were used to deplete SMYD5. The pLKO. I vector containing a scrambled shRNA was used as control. Pseudotyped viral stocks were produced in 2×106 HEK293T cells by the calcium phosphate method by co-transfecting 10 μg of shRNA-expressing lentiviral vectors, with 6.5 μg of the lentiviral packaging construct pCMVdelta R8.91 and 3.5 μg of VSV-G glycoprotein-expressing vector (Naldini et al. Science 1996; 272:263-7), and titered for p24 content. J-Lat 5A8, A72 and A2 cells were spininfected with virus (1 ng of p24 per 106 cells) containing shRNAs against KMTs or nontargeting control shRNAs for 2 hr. Infected cells were selected with puromycin (2 μg/ml; Sigma-Aldrich) and after 4 days of selection, cells were treated with the indicated concentration of drugs. The percentage of GFP+ cells was determined after 18 h using a MACSQuant VYB FACS analyzer (Miltenyi Biotech GmbH). Cell viability was monitored by forward-and-side scatter analysis. Analysis was conducted on 3×10,000 live cells per condition. Data were analyzed using FlowJo 9.5 (Tree Star).
RNA Isolation, Reverse Transcription, and Quantitative RT-PCR
RNA was isolated using RNeasy Plus Mini Kit (Qiagen) and reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) as per the manufacturer's instructions. Quantitative RT-PCR was carried out using Maxima SYBR Green qPCR Master Mix (Thermo Scientific) on SDS 2.4 software (Applied Biosystems) in a total volume of 12 μL. Primer efficiencies were around 100%. Dissociation curve analysis was performed after the end of the PCR to confirm the presence of a single and specific product.
In Vitro Methylation Assays
Methylation assays were performed as described (Nishioka et al. Mol Cell 2002; 9:1201-13). For reactions, 2 μg of histones (isolated from HEK293T cells), or synthetic Tat peptides (German Cancer Research Center) were incubated with recombinant full-length SMYD5 (Active Motif, #31409, purified from Sf9 cells) in a buffer containing 50 mM Tris-HCl, pH 9, 0.01% Tween 20, 2 mM DTT and 1.1 μCi of H3-labeled SAM (Perkin Elmer) overnight at 30° C. Reaction mixtures were fractionated on 15% SDS-PAGE for proteins or on 10-20% Tris-Tricine gradient gels for peptides (BioRad). After Coomassie staining, gels were treated with Amplify (GE Healthcare) for 30 min, dried and exposed to hyperfilm (GE Healthcare) overnight.
Luciferase Assays
1×105 HeLa cells were transfected with 25 ng LTR-Luciferase construct and 50, 100 or 500 ng ng of DNA containing SMYD5 expressing plasmids or empty vector using X-tremegene 9 following manufacturer instructions (Roche Diagnostics, Indianapolis, Ind.). Cells were harvested 48 hr after stimulation, washed one time with PBS, and lysed in 60 μl of Passive Lysis Buffer (Dual-Luciferase Assay System-Promega). After 15 min of lysis, the luciferase activity in cell extracts was quantified with a Monolight 2010 Luminometer (Analytical Luminescence Laboratory) after mixing 20 μl of lysate with 100 μl of substrate. Relative light units (RLU) were normalized to protein content determined by Bradford assay (BioRad). Co-transfection of 10 ng eF1α-Renilla was used to control for transfection efficiency.
Results
To confirm results from the shRNA screen, SMYD5 was individually knocked down in J-Lat 5A8 cells. Cells were transduced with lentiviral vectors expressing two different shRNAs targeting SMYD5 or a scrambled control shRNA, followed by puromycin treatment to select successfully transduced cells. The shRNA knockdown was confirmed using qPCR and failed for #1 and was ˜50% effective for #2 (
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
This application is a continuation of U.S. patent application Ser. No. 16/422,753, filed May 24, 2019, which application is a division of U.S. patent application Ser. No. 15/488,212, filed Apr. 14, 2017, issued on Jul. 16, 2019 as U.S. Pat. No. 10,351,853, which application is a Continuation-In-Part of International Application No. PCT/US2015/055377, filed Oct. 13, 2015, which application claims the benefit of priority to U.S. Provisional Application No. 62/063,822, filed Oct. 14, 2014, which applications are incorporated herein by reference in their entirety.
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| 20220090085 A1 | Mar 2022 | US |
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| Parent | 15488212 | Apr 2017 | US |
| Child | 16422753 | US |
| Number | Date | Country | |
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| Parent | 16422753 | May 2019 | US |
| Child | 17380773 | US |
| Number | Date | Country | |
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| Parent | PCT/US2015/055377 | Oct 2015 | US |
| Child | 15488212 | US |
