Patent Application
20220073495
The instant application contains a Sequence Listing which has been submitted via ASCII copy created on Mar. 18, 2021, referred to as ‘106546-698161_Seq_Listing_ST25.txt’ having 75 sequences.
The present disclosure generally relates to influenza treatment or prevention. In particular, the present disclosure relates to compounds that prevent influenza virus replication through inhibition of influenza viral mRNA nuclear export, as well as methods for treating or preventing influenza using the compounds.
Influenza virus is a major human pathogen that kills approximately 500,000 people worldwide every year and between 15,000 to 40,000 Americans yearly, depending on the influenza strain. The 1918 influenza pandemic killed approximately 50 million people worldwide. Currently available prevention measures and treatments include vaccines and a few antiviral drugs. However, these treatment methods are limited by the mutability of the virus and the development of resistance. As antiviral treatments currently approved for clinical use target viral proteins directly, such treatments have an increased probability of developing strains resistant to these antiviral compositions.
Additionally, antiviral drugs are largely only effective if administered in the first 48 hours following infection and vaccines are less effective in treating elderly populations. Accordingly, in view of the lack of robust and diverse medical interventions available, additional antiviral compositions, methods, and therapeutic strategies for the treatment or prevention of influenza are desirable. The present disclosure fulfills this long standing need.
The present disclosure provides, in part, identification of compounds that inhibit influenza viral M mRNA processing and nuclear export and are useful in treatment or prevention of an influenza viral infection. The identified compounds target cellular proteins instead of viral proteins. The present disclosure further provides methods of treating or preventing an influenza viral infection using the compounds identified herein and kits comprising the same.
Accordingly, one aspect of the present disclosure provides a method of treating an influenza viral infection in a subject in need thereof. Such method comprises administering to the subject a therapeutically effective amount of an inhibitor of influenza viral M mRNA nuclear export. In one embodiment, the inhibitor targets a cellular protein in viral M mRNA speckle-export pathway. By way of non-limiting example, the cellular protein is a binding partner of viral NS1 protein.
In one embodiment, the inhibitor is a compound comprising a structural formula selected from the group consisting of Structural Formula I, Structural Formula II, Structural Formula III, Structural Formula IV, Structural Formula V, Structural Formula VI, Structural Formula VII, Structural Formula VIII, Structural Formula IX, Structural Formula X, Structural Formula XI, Structural Formula XII, Structural Formula XIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, Structural Formula XIX, Structural Formula XX, Structural Formula XXI, Structural Formula XXII, Structural Formula XXIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, and Structural Formula XXIX, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula I below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof; wherein R1 is an unsubstituted or substituted aryl or heteroaryl; R2 and R3 are either the same or different and are selected from H or alkyl; X is selected from NH, NR5, O, and S; R4 is appended to an optional ring as part of a benzo-fused heteroaryl and is selected from H, alkyl or halogen; and R5 is an alkyl or aryl.
In one embodiment, the inhibitor is a compound comprising Structural Formula II below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula III below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula IV below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
Another aspect of the present disclosure provides a method of preventing an influenza viral infection in a subject in need thereof. Such method comprises administering to the subject a therapeutically effective amount of an inhibitor of influenza viral M mRNA nuclear export. In one embodiment, the inhibitor targets a cellular protein in viral M mRNA speckle-export pathway. By way of non-limiting example, the cellular protein is a binding partner of viral NS1 protein.
In one embodiment, the inhibitor is a compound comprising a structural formula selected from the group consisting of Structural Formula I, Structural Formula II, Structural Formula III, Structural Formula IV, Structural Formula V, Structural Formula VI, Structural Formula VII, Structural Formula VIII, Structural Formula IX, Structural Formula X, Structural Formula XI, Structural Formula XII, Structural Formula XIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, Structural Formula XIX, Structural Formula XX, Structural Formula XXI, Structural Formula XXII, Structural Formula XXIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, and Structural Formula XXIX, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula I below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof; wherein R1 is an unsubstituted or substituted aryl or heteroaryl; R2 and R3 are either the same or different and are selected from H or alkyl; X is selected from NH, NR5, O, and S; R4 is appended to an optional ring as part of a benzo-fused heteroaryl and is selected from H, alkyl or halogen; and R5 is an alkyl or aryl.
In one embodiment, the inhibitor is a compound comprising Structural Formula II below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula III below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula IV below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
Still another aspect of the present disclosure provides a kit for treating or preventing an influenza viral infection. Such kit comprises a therapeutically effective amount of an inhibitor of influenza viral M mRNA nuclear export, a means of administering the inhibitor, and instructions for use.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In order to describe the manner in which the advantages and features of the disclosure can be obtained, reference is made to embodiments thereof which are illustrated in the appended drawings. It is to be understood that these drawings depict only exemplary embodiments of the disclosure and are not therefore to be considered to be limiting of its scope, the principles herein are described and explained with additional specificity and detail through the use of the accompanying drawings.
It will be appreciated that numerous specific details are set forth in order to provide a thorough understanding of the embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein can be practiced without these specific details. In other instances, methods, procedures and components have not been described in detail so as not to obscure the related relevant feature being described. Also, the description is not to be considered as limiting the scope of the embodiments described herein.
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.
“About” is used herein to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
As used herein, the term “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).
As used herein, the transitional phrase “consisting essentially of” (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Thus, the term “consisting essentially of” as used herein should not be interpreted as equivalent to “comprising.”
Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
As used herein, “treatment,” “therapy” and/or “therapy regimen” refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and/or the remission of the disease, disorder or condition.
As used herein, the term “inhibit” or “inhibiting” refers to reduction in the amount, levels, density, turnover, association, dissociation, activity, signaling, or any other feature associated with the protein.
As used herein, “administration” of a disclosed compound encompasses the delivery to a subject of a compound as described herein, or a prodrug or other pharmaceutically acceptable derivative thereof, using any suitable formulation or route of administration, as discussed herein.
The term “effective amount” or “therapeutically effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results including, but not limited to, disease treatment, as illustrated below. In some embodiments, the amount is that effective for detectable reduction of pain. In some embodiments, the amount is that effective for alleviating, reducing or eliminating a pathologic pain.
The therapeutically effective amount can vary depending upon the intended application, or the subject and disease condition being treated, e.g., the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the weight and age of the patient, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of cell migration. The specific dose will vary depending on, e.g., the particular compounds chosen, the species of subject and their age/existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
As used herein, the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The methods and compositions disclosed herein can be used on a sample either in vitro (for example, on isolated cells or tissues) or in vivo in a subject (i.e. living organism, such as a patient). In some embodiments, the subject comprises a subject suffering from an influenza viral infection.
The present disclosure illustrates that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, thus providing an avenue to preferentially target influenza virus. Through a high-content, image-based chemical screen, inhibitors of viral mRNA biogenesis and nuclear export were identified that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.
The present disclosure provides methods and compositions for the treatment or prevention of influenza. In particular, the present disclosure provides compounds that prevent influenza virus replication through inhibition of influenza viral mRNA nuclear export, as well as methods for the treatment or prevention of influenza that include administration of such compounds.
According to one aspect of the present disclosure, there is provided a method of treating an influenza viral infection in a subject in need thereof. Such method comprises administering to the subject a therapeutically effective amount of an inhibitor of influenza viral M mRNA nuclear export.
In one embodiment, the inhibitor targets a cellular protein in viral M mRNA speckle-export pathway. By way of non-limiting example, the cellular protein is a binding partner of viral NS1 protein. By way of non-limiting example, the cellular protein targeted by the inhibitor is NS1-BP.
Since Influenza A viruses are human pathogens with limited therapeutic options, it is crucial to devise strategies for the identification of new classes of antiviral medications. The Influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Nuclear speckles are known to be storage sites for splicing and other RNA processing factors, and this process requires key viral-host interactions for both splicing and nuclear export of the viral M2 mRNA. This suggests a pathway in which the viral NS1 protein interacts with the cellular NS1-BP protein, which in turn binds hnRNP K to target the M1 mRNA from the nucleoplasm to nuclear speckles. At this nuclear body, the U1 snRNP and/or dissociation of NS1 induces a remodeling of the protein-RNA complex in a manner that hnRNP K recruits U1 snRNP to the M2 5′ splice site on M1 mRNA to mediate splicing. Then, NS1 and NS1-BP together with key members of the mRNA nuclear export machinery (the RNA helicase UAP56 and the mRNA export factor Aly/REF) promote nuclear export of M1 and M2 mRNAs.
Since this splicing-export pathway through nuclear speckles does not impact bulk mRNA but only a subset of viral and cellular mRNAs, chemical compounds antagonizing this process would have the potential of not being overly toxic and could inhibit virus replication. Through an image-based chemical screen using single-molecule RNA-FISH to detect the viral M mRNA (M1 and M2 mRNAs), chemical compounds were identified that would inhibit different steps of this speckle-export intranuclear pathway yet would not significantly compromise bulk poly(A) RNA.
Accordingly, in one embodiment, the inhibitor is a compound comprising a structural formula selected from the group consisting of Structural Formula I, Structural Formula II, Structural Formula III, Structural Formula IV, Structural Formula V, Structural Formula VI, Structural Formula VII, Structural Formula VIII, Structural Formula IX, Structural Formula X, Structural Formula XI, Structural Formula XII, Structural Formula XIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, Structural Formula XIX, Structural Formula XX, Structural Formula XXI, Structural Formula XXII, Structural Formula XXIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, and Structural Formula XXIX, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula I below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof; wherein R1 is an unsubstituted or substituted aryl or heteroaryl; R2 and R3 are either the same or different and are selected from H or alkyl; X is selected from NH, NR5, O, and S; R4 is appended to an optional ring as part of a benzo-fused heteroaryl and is selected from H, alkyl or halogen; and R5 is an alkyl or aryl.
In one embodiment, the inhibitor is a compound comprising Structural Formula II below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is BrC1=CC═C(NC(═O)CSC2=NC3=C(N2)C═CC=C3)N=C1
In one embodiment, the inhibitor is a compound comprising Structural Formula III below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is CN1C(SCC(═O )NC2=CC═C(Br)C=N2)=NC2=CC═CC=C12.
In one embodiment, the inhibitor is a compound comprising Structural Formula IV below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is CC1=C2N═C(NC2=CC=C1)SCC(═O)NC1=CC═C(Br)C=N1.
The method disclosed above and herein can be used to treat an influenza viral infection caused by different influenza viruses. In one embodiment, the influenza viral infection may be caused by an influenza A virus (IAV). By way of non-limiting example, the influenza virus A is subtype H1N1, H2N2, H3N2, or H5N1. In one embodiment, the influenza viral infection may be caused by an influenza B virus (IBV).
In one embodiment, at least one additional therapeutic agent may be further administered to the subject in need thereof. By way of non-limiting example, the additional therapeutic agent may be Rapivab, Relenza, Tamiflu, Xofluza, or a combination thereof.
When used to treat or prevent such diseases (e.g., flu), the compounds described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases. In one embodiment, a compound comprising the Structural Formula II may be administered together with a compound comprising Structural Formula III and/or a compound comprising Structural Formula IV. The compounds may also be administered in mixture or in combination with agents useful to treat other disorders or maladies. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.
In one embodiment, the compounds may be administered in combination with a therapeutic treatment modality. By way of non-limiting example, the therapeutic treatment modality may be a flu vaccine.
Pharmaceutical compositions comprising the compound(s) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically. The exact nature of the carrier, diluent, excipient or auxiliary will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use. The composition may optionally include one or more additional compounds.
The compounds may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.
In one embodiment, the compounds described above and herein may be administered orally, buccally, sublingually, rectally, intravenously, intramuscularly, topically, auricularly, conjunctivally, nasally, via inhalation, or subcutaneously.
Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.
For topical administration, the compound(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.
For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films or enteric coatings.
Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophore™ or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the compound, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.
For nasal administration or administration by inhalation or insufflation, the compound(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
For ocular administration, the compound(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.
For prolonged delivery, the compound(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The compound(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compound(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compound(s).
Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compound(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.
The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The compound(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or slowing the progression of the disease, regardless of whether improvement is realized.
The amount of compound(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular compound(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.
Determination of an effective dosage of compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC50 of the particular compound as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.
Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and/or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and/or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.
Another aspect of the present disclosure provides a method of preventing an influenza viral infection in a subject in need thereof. Such method comprises administering to the subject a therapeutically effective amount of an inhibitor of influenza viral M mRNA nuclear export. In one embodiment, the inhibitor targets a cellular protein in viral M mRNA speckle-export pathway. By way of non-limiting example, the cellular protein is a binding partner of viral NS1 protein. By way of non-limiting example, the cellular protein is NS1-BP.
In one embodiment, the inhibitor is a compound comprising a structural formula selected from the group consisting of Structural Formula I, Structural Formula II, Structural Formula III, Structural Formula IV, Structural Formula V, Structural Formula VI, Structural Formula VII, Structural Formula VIII, Structural Formula IX, Structural Formula X, Structural Formula XI, Structural Formula XII, Structural Formula XIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, Structural Formula XIX, Structural Formula XX, Structural Formula XXI, Structural Formula XXII, Structural Formula XXIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, and Structural Formula XXIX, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula I below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof; wherein R1 is an unsubstituted or substituted aryl or heteroaryl; R2 and R3 are either the same or different and are selected from H or alkyl; X is selected from NH, NR5, O, and S; R4 is appended to an optional ring as part of a benzo-fused heteroaryl and is selected from H, alkyl or halogen; and R5 is an alkyl or aryl.
In one embodiment, the inhibitor is a compound comprising Structural Formula II below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is BrC1=CC═C(NC(═O)CSC2=NC3=C(N2)C═CC=C3)N=C1.
In one embodiment, the inhibitor is a compound comprising Structural Formula III below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is CN1C(SCC(═)NC2=CC═C(Br)C=N2)=NC2=CC═CC=C12.
In one embodiment, the inhibitor is a compound comprising Structural Formula IV below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is CC1=C2N═C(NC2=CC=C1)SCC(═O)NC1=CC═C(Br)C=N1.
The method disclosed above and herein can be used to prevent an influenza viral infection caused by different influenza viruses. In one embodiment, the influenza viral infection may be caused by an influenza A virus (IAV). By way of non-limiting example, the influenza virus A is subtype H1N1, H2N2, H3N2, or H5N1. In one embodiment, the influenza viral infection may be caused by an influenza B virus (IBV).
In one embodiment, at least one additional therapeutic agent may be further administered to the subject in need thereof. By way of non-limiting example, the additional therapeutic agent may be Rapivab, Relenza, Tamiflu, Xofluza, or a combination thereof.
When used to treat or prevent such diseases (e.g., flu), the compounds described herein may be administered singly, as mixtures of one or more compounds or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases. The compounds may also be administered in mixture or in combination with agents useful to treat other disorders or maladies. The compounds may be administered in the form of compounds per se, or as pharmaceutical compositions comprising a compound.
In one embodiment, the compounds may be administered in combination with a therapeutic treatment modality. By way of non-limiting example, the therapeutic treatment modality may be a flu vaccine.
In one embodiment, the inhibitor or at least one additional therapeutic agent is administered orally, buccally, sublingually, rectally, intravenously, intramuscularly, topically, auricularly, conjunctivally, nasally, via inhalation, or subcutaneously.
According to still another aspect of the present disclosure, there is provided a kit for treating or preventing an influenza viral infection. Such kit comprises a therapeutically effective amount of an inhibitor of influenza viral M mRNA nuclear export, a means of administering the inhibitor, and instructions for use.
In one embodiment, the inhibitor is a compound comprising a structural formula selected from the group consisting of Structural Formula I, Structural Formula II, Structural Formula III, Structural Formula IV, Structural Formula V, Structural Formula VI, Structural Formula VII, Structural Formula VIII, Structural Formula IX, Structural Formula X, Structural Formula XI, Structural Formula XII, Structural Formula XIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, Structural Formula XIX, Structural Formula XX, Structural Formula XXI, Structural Formula XXII, Structural Formula XXIII, Structural Formula XIV, Structural Formula XV, Structural Formula XVI, Structural Formula XVII, Structural Formula XVIII, and Structural Formula XXIX, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
In one embodiment, the inhibitor is a compound comprising Structural Formula I below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof; wherein R1 is an unsubstituted or substituted aryl or heteroaryl; R2 and R3 are either the same or different and are selected from H or alkyl; X is selected from NH, NR5, O, and S; R4 is appended to an optional ring as part of a benzo-fused heteroaryl and is selected from H, alkyl or halogen; and R5 is an alkyl or aryl.
In one embodiment, the inhibitor is a compound comprising Structural Formula II below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is BrC1=CC═C(NC(═O)CSC2=NC3=C(N2)C═CC=C3)N=C1.
In one embodiment, the inhibitor is a compound comprising Structural Formula III below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is CN1C(SCC(═O)NC2=CC═C(Br)C=N2)=NC2=CC═CC=C12.
In one embodiment, the inhibitor is a compound comprising Structural Formula IV below:
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, or derivative thereof.
By way of non-limiting example, the inhibitor is CC1=C2N═C(NC2=CC=C1)SCC(═O)NC1=CC═C(Br)C=N1.
In one embodiment, the kit further comprises at least one additional therapeutic agent. By way of non-limiting example, the additional therapeutic agent may be Rapivab, Relenza, Tamiflu, Xofluza, or a combination thereof.
In some embodiments, the kit is packaged in a container with a label affixed to the container or included in the package that describes use of the compounds described herein. Exemplary containers include, but are not limited to, a vessel, vial, tube, ampoule, bottle, flask, and the like. It is contemplated that the container is made from material well-known in the art, including, but not limited to, glass, polypropylene, polystyrene, and other plastics. In various aspects, the compounds are packaged in a unit dosage form. In various aspects, the kit contains a label and/or instructions that describes use of the contents for pain treatment.
The following Examples are provided by way of illustration and not by way of limitation.
Human lung adenocarcinoma epithelial cells (A549) and MDCK cells, obtained from ATCC (American Type Culture Collection), were maintained in high-glucose DMEM (Gibco), 10% FBS (Sigma), and 100 units/mL Pen/Strep antibiotics at 37° C. with 5% CO2. Primary human bronchial epithelial cells were cultured as previously reported (Peters-Hall, et al., 2019, The FASEB Journal, 00: 1-13). A549 cells stably expressing UAP56 E179A mutant were generated according to Hondele et al. (2019, Nature 573(7772): 144-8).
Transfections and siRNAs
siRNAs were reverse transfected with A549 cells using RNAiMAX (Invitrogen) in OptiMEM (ThermoFisher) by the manufacturer's instructions. After 24 h transfection, media was replaced with growth media. Knockdown was allowed to continue for 48 h before compound treatment or infections occurred. siRNAs used include UAP56 and MISSION siRNA Universal Negative Control #2 (Sigma-Aldrich), ON-TARGETplus siRNAs against SON and ON-TARGETplus Non-targeting Control #2 (Dharmacon, ThermoFisher), and 3′UTR siUAP56 sequence 5′-GCUUCCAUCUUUUGCAUCAUU-3′ (SEQ ID NO: 73) (Dharmacon).
The NS1-BP gene was knocked out in A549 cells by genome editing using CRISPR-Cas9. In brief, the genomic target oligos (Forward: CACCGTGCTTATGGCCATTCTCACG (SEQ ID NO: 74), Reverse: AAACCGTGAGAATGGCCATAAGCAC (SEQ ID NO: 75)) were cloned into a lentiCRISPRv2 vector. The plasmid was co-transfected into HEK293T cells, obtained from ATCC (American Type Culture Collection), with the packaging plasmids pVSVg and psPAX2, generating lentivirus to infect A549 cells. Then, cells were clonally selected using puromycin (1.0 μg/ml) for 7 days followed by 3 days without selection for expansion. Clones were isolated and expanded to generate lysates for western blot analysis using anti-NS1-BP antibody. Candidate clones were subjected to genomic sequencing using amplicons flanking the sgRNA-target site. Growth rates were determined by measuring ATP levels. Cells were tested at 24 h, 48 h, 72 h, and 96 h after plating equal number of NS1-BP+/+ and NS1-BP31/− cells. ATP was measured by luminescence using CellTiter-Glo (Promega) according to the manufacturer's instructions.
Influenza A viruses (A/WSN/33, A/Vietnam/1203/04, A/Panama/99) were generated in embryonated eggs or in MDCK cells after growth from a clonal population of virus at low multiplicity of infection to avoid accumulation of defective virus particles. In MDCK cells, virus was amplified at MOI 0.1-0.001 in infection media containing EMEM (ATCC, 30-2003), 10 mM HEPES (Gibco), 0.125% BSA (Gibco), 0.5 μg/mL TPCK trypsin (Worthington Biomedical Corporation). Cells were incubated with virus for 1 hour at 37° C., then washed before amplification in infection media. After cell death was observed at 48-72 hours post-infection, supernatants were centrifuged at 1,000×g for 10 minutes to remove cell debris, aliquoted, and stored at −80° C. All virus stocks are controlled for an appropriate ratio of HA/PFU titer and sequenced by RNAseq to confirm the full sequence of the virus.
A549 cells were infected with A/WSN/33 and A/Vietnam/1203/04 at MOI 0.01, or with A/Panama/99 at MOI 0.1 in the absence or presence of compound 2 at concentrations depicted in the figures. Supernatants were collected from each condition 24 h post-infection and viral particles were tittered by plaque assay as following: MDCK cells were seeded in 6-well plates to reach confluency the next day. At confluency, ten-fold serial dilutions of each sample's supernatant were diluted in PBS containing 100 units/mL Pen/Strep antibiotics, 0.2% BSA, 0.9 mM CaCl2, and 1.05 mM MgCl2. After infection with each dilution, cells were overlaid with a 1:1 mixture of 2×-15 media and 2% Oxiod Agar (Final concentration of 1×L-15 media and 1% Agar). Plaques formed at 24 h for A/WSN/33 and A/Vietnam/1203/04, or 48 h for A/Panama/99 were counted and titers determined. Primary human bronchial epithelial cells were infected with A/WSN/33 at MOI 0.1 for 24 h in the absence or presence of compound 2 at depicted concentrations. Supernatants were subjected to plaque assays as described above. Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer's instructions, concurrent with viral replication assay.
smRNA-FISH
smRNA-FISH was performed as previously described (Mor et al., 2016, Nat Microbiol. 1(7): 16069), which includes the sequences of M1 and NS1 probes except for the HA probes that are listed in Table 1 below. Briefly, cells were grown on glass coverslips (Fisherbrand, FisherScientific) coated with 1mL of 0.1% gelatin (Sigma-Aldrich). Cells were fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences) in PBS for 15 min before incubation in 70% ethanol for 12 h at 4° C. Coverslips were then placed in wash buffer for 5 min, containing Nuclease Free Water, 2×SSC Buffer (Sigma), and 10% formamide (Sigma). The coverslips where then removed and incubated in hybridization buffer containing FISH probe. Hybridization occurred at 37° C. for 4 h, then cells were washed with wash buffer for 30 min at 37° C. Coverslips were then washed twice for 5 min in PBS and stained with 1 μg/ml Hoechst 33258 (Molecular Probes/Life Technologies) for 10 min. Coverslips were briefly washed with PBS before mounting in ProLong Gold antifade reagent (Life Technologies).
Table 1 lists the forty-eight, 20 nt DNA probes labeled with Quasar 570 (BIOSEARCH TECHNOLOGIES) that were designed to hybridize with the Influenza WSN full length HA mRNA.
To identify chemical inhibitors of viral M mRNA processing and nuclear export, A549 cells were treated with 232,500 chemical compounds available from the University of Texas Southwestern Medical Center High Throughput Screening core facility. Cells were treated with 2.5 μM compound for 30 minutes and incubated at 37° C. in 5% CO2. Cells were then infected with influenza A/WSN/33 virus at MOI of 2 and returned to incubation as before. At 7.5 hours post-infection, cells were fixed with 4% paraformaldehyde and subjected to RNA-FISH. To localize M mRNA, forty-five FISH probes labeled with Quasar 570 were used that cover the entire M mRNA segment, as previously reported (Mor et al., 2016, Nat Microbiol. 1(7): 16069). Nuclei were stained with 1 μg/m1 Hoechst 33342 dye. M mRNA distribution between the nucleus and the cytoplasm was detected using the IN Cell Analyzer 6000 (GE Healthcare, Marlborough MA). Multiple fields per well were taken at 20× magnification using the Hoechst and dsRed widefield fluorescence filters. Image analysis was performed in a GE IN Cell Analyzer Workstation 3.7.3 (GE Healthcare) using the multi-target analysis template. Individual nuclei were segmented using a top-hat filter on the Hoechst channel with the default sensitivity setting. For samples detecting the M1 mRNA, the cell body was segmented using the region growing method on the M1 mRNA channel. This method uses the nuclei as the seed and then expands outwards until the edge of the stain is reached. For samples detecting poly(A) RNA, the poly(A) RNA channel was instead used to define the cell body region using the region growing method. For each segmented nucleus and cell pair, the mean and total signal intensities of the nuclear and cytoplasmic chambers were calculated for the poly(A) RNA (where applicable) and M1 mRNA channels. The mean nuclear to mean cytoplasmic (N/C) ratio was then calculated for both mRNA probes for each cell. Finally, the average N/C ratios per well were calculated and used for hit identification. The results were imported into the GeneData Screener™ (Basel, Switzerland; version 13.0.6) software analysis suite to normalize and summarize the overall M mRNA intensity as well as nuclear to cytosolic ratio in terms of a Z-score as previously described (Zhang et al., 2015, Science 348: 6240; Wu et al., 2008, Journal of Biomolecular Screening, 13(2): 159-67).
In the primary screen, compounds with a robust Z-score of less than −3 for intensity were considered hits affecting virus replication. Compounds with a Z-score greater than 3 in the nuclear/cytosolic ratio were selected as hits for inhibition of nuclear export. Any compound that lowered the nuclear count to a Z-score of −3 or lower was considered cytotoxic and not included in follow-up experiments. Compounds (1,125) that had the highest activity were selected for confirmation and retested in triplicate at a compound concentration of 2.5 μM. All imaging confirmation and follow-up assays included a bulk poly(A) RNA probe linked to Quasar 670 for FISH imaging. As with the M mRNA probe, total intensity and N/C ratio were also measured for the poly(A) RNA probe. The 600 compounds with the highest activity from the confirmation assay were subjected to 12-point dose response curves ranging from 0.5 nM to 50 μM at 0.5 log dose intervals. Of the 600 compounds tested, 413 compounds had a measureable effect on bulk poly(A) RNA and were excluded from further testing. The remaining 187 compounds that inhibited viral mRNA nuclear export and/or decreased viral mRNA levels but had no substantial effect on the host cell poly(A) RNA were categorized into 3 major phenotypes. These include 22 compounds that retained viral M mRNA in the nucleus, 33 compounds that decreased viral M mRNA levels, and 132 compounds that decreased overall levels and inhibited nuclear export of viral M mRNA. Clustering analysis of confirmed hits was performed with Pipeline Pilot v16 (Biovia, Inc.) using ECFP4 fingerprints (Rogers and Hahn, 2010, J Chem Inf Model, 50(5):742-54).
Total cell fluorescence intensity or fluorescence intensity in the nucleus and cytoplasm analysis was conducted. Images deconvolved with AutoQuant software were analyzed using Imaris (Bitplane). The Surfaces tool was used to segment fluorescence within the cytoplasm and nucleus of each cell quantified. Statistical analyses for imaging studies and qPCR data in the figures mentioned above were performed using the two-sample, two-tailed, t-test.
Compound 2-thiobenzimidazole was initially purchased from TimTec (HTS04595) as well as synthesized in-house. Comparative compound JMN3-003 was synthesized as previously described (Moore et al., 2013, J Org. Chem. 9: 197-203). All compounds were dissolved in dimethylsulfoxide (DMSO). Compound 2 was synthesized and characterized as following:
2-((1H-benzo[d]imidazol-2-yl)thio)-N-(5-bromopyridin-2-yl)acetamide
A mixture of 2-mercaptobenzimidazole (30.0 mg, 0.2 mmol, 1.0 equiv.) and crushed potassium hydroxide (11.2 mg, 0.2 mmol, 1.0 equiv.) in 2 ml of ethanol was kept at reflux for 2 hours. The reaction mixture was cooled down to room temperature, N-(5-bromopyridin-2-yl)-2-chloroacetamide (49.9 mg, 0.2 mmol, 1.0 equiv.) was added, and the reaction was stirred for overnight. The resulting reaction mixture was concentrated under reduced pressure. 2.0 ml of saturated ammonium chloride solution and 2.0 ml of dichloromethane were added to the residue. The organic layer was separated, washed with 2.0 ml of brine, then dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude was further purified by silica gel chromatography using 60% of ethyl acetate in hexane to afford 54 mg white solid as product, yield 74%.
δppm 8.31-8.24 (m, 1H), 8.03 (d, J=8.8 Hz, 1H), 7.72 (ddd, J=8.9, 2.8, 1.5 Hz, 1H), 7.48 (br, 2H), 7.20-7.08 (m, 3H), 4.03 (s, 2H).
δppm 168.23, 149.92, 149.48, 148.78, 140.57, 122.95, 122.35, 115.53, 114.86, 109.97, 36.24.
MS (ESI) m/z=363.0 ([M+H]+), C14H11BrN4OS requires 363.0.
ATP was measured by luminescence using the CellTiter-Glo kit (Promega) according to the manufacturer's instructions.
Total RNA was isolated from A549 cells using the RNeasy Plus Mini Kit (Qiagen) and reverse transcribed into cDNA by SuperScript II reverse transcriptase (Invitrogen), each according to the manufacturers' protocols. Samples were then amplified in a LightCycler 480 quantitative real-time PCR (qPCR) system (Roche) using SYBR Green I (Roche) and sequence specific primers. RT-PCR Primer Sequences are listed below:
Cells were treated with 0.1% DMSO or 2.5 μM compound 2 for 9 hours. Nuclear and cytoplasmic fractions were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Controls are discussed in Table 3. Total RNA was isolated total cell lysates, nuclear and cytoplasmic fractions using the RNeasy Plus Mini Kit (Qiagen). RNA samples were then analyzed in the Agilent 2100 Bioanalyzer to determine RNA quality (RIN Score 8 or higher). RNA concentration was determined using the Qubit fluorometer. A TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina) was used to prepare 4 μg of DNAse-treated RNA for poly(A) RNA purification and fragmentation before strand specific cDNA synthesis. cDNA libraries were a-tailed and ligated to indexed adapters. Samples were then PCR amplified and purified with Ampure XP beads and validated with the Agilent 2100 Bioanalyzer. Samples were quantified again by Qubit before being normalized and pooled to be ran on the Illumina HiSeq 2500 using SBS v3 reagents. Raw FASTQ files were analyzed using FastQC v0.11.2 (Andrews S. 2010, FastQC: A Quality Control Tool for High Throughput Sequence Data) and FastQ Screen v0.4.4 (Wingett and Andrews, PubMed PMID: 30254741) and reads were quality-trimmed using fastq-mcf (ea-utils/1.1.2-806). The trimmed reads were mapped to the hg19 assembly of the human genome (the University of California, Santa Cruz, version from igenomes) using STAR v2.5.3a (Dobin et al., PubMed PMID: 23104886). Duplicated reads were marked using Picard tools (v1.127; Broad Institute), the RNA counts generated from FeatureCounts (Liao et al., 2014, Bioinformatics 30(7): 923-30) were TMM normalized, and differential expression analysis was performed using edgeR (Robinson et al., 2010, Bioinformatics 26(1): 139-40). Expression data is represented as TPM (Transcripts per Million). Genes with mRNA TPM values of zero in either the control or experiment conditions were removed from the analysis. Log2 of the average TPM values for the remaining genes of each condition (total, nuclear, and cytoplasmic) were calculated. Only mRNAs with Log2TPM>−1 were considered for further analysis to remove experimental background noise. The TPM readings of the experiment compared with control samples were used to calculate the positive and negative fold changes from their ratios. The differentially expressed mRNAs with fold changes of + or −1.5 FC were subjected to GSEA to obtain the enriched pathways.
Pathway and network analysis were conducted using Gene Set Enrichment Analysis (GSEA) (Subramanian et at., 2005, PNAS USA 102(43): 15545-50) software and the functional datasets were CP: Canonical pathways from the MSigDB (Liberzon et al., 2015, Cell Syst. 1(6): 417-25; Liberzon et al., 2011, Bioinformatics 27(12): 1739-40).
Cell lysis was performed in 250 mM Tris HCl pH 6.8, 40% Glycerol, and 8% SDS. Western blot was performed as previously described (Tsai et al., 2013, PLoS pathogens 9(6): e1003460). Antibodies used in this study to detect viral proteins include Influenza A virions (Meridian Life Science B65141G), M1 and M2 (Thermo MA1-082), NA (GeneTex GTX125974), PA (GeneTex GTX118991), PB1 (Santa Cruz sc-17601), PB2 (Santa Cruz sc-17603), and NS1 (a gift from J.A. Richt, National Animal Disease Center, Iowa) (Solorzano et al., 2005, Journal of virology 79(12): 7535-43). Antibodies against cellular proteins include β-actin (Sigma A5441) and UAP56 [Anti-BAT1 (C-TERMINAL antibody produced in rabbit, Millipore SAB1307254). Horseradish peroxidase (HRP)-conjugated secondary antibodies include donkey anti-rabbit, sheep anti-mouse (GE Healthcare NA934V and NA931V, respectively), and donkey anti-goat (Jackson Immunoresearch 705-035003). Quantification of protein band intensity was performed using Image Studio software (LI-COR Imaging). Each protein band was normalized to its corresponding loading control. Values listed below each band represent relative band intensity to its corresponding control.
Knockdown of the cellular NS1-BP protein was previously reported to inhibit influenza virus M mRNA splicing and nuclear export through host nuclear speckles (Mor et al., 2016, Nat Microbiol. 1(7): 16069). In this study, NS1-BP was knocked out using the CRISPR/Cas9 system (
Next, a high-throughput screening was performed to select inhibitors of viral M mRNA processing and nuclear export. The previously reported protocol to visualize the M mRNAs during virus infection (Mor et al., 2016, Nat Microbiol. 1(7): 16069) was adapted and a high-throughput screening assay was designed to identify compounds that alter M mRNA expression and trafficking without significantly compromising bulk cellular poly(A) RNA levels or intracellular distribution. The high throughput screen was performed using a chemical library of 232,500 compounds. As shown in
The identified inhibitors of viral mRNA nuclear export are provided in Table 2. As shown therein, compounds 1-28, corresponding to structural formulas III, II and IV-XXIX, were identified through the above screening as compounds that inhibit nuclear export of influenza virus mRNAs and consequently prevent influenza virus replication at non-toxic concentrations. In Table 2, virus replication was assessed in A549 cells, which were infected with A/WSN/33 at MOI 0.01 in the absence or presence of compound 2 at different concentrations. After 24 h post infection, cells were fixed with 4% formaldehyde for 30 min. Cells were briefly washed with PBS, then permeabilized with 0.1% Triton X-100 in PBS for 15 minutes. Blocking occurred at room temperature for 1 hour with 0.5% BSA in PBS followed by incubation with the NP antibody (HT103) in 0.5% BSA in PBS for 1 h at room temperature. Cells were washed with PBS 2× and incubated with a fluorescently-labeled secondary antibody, alexa-fluor-488 (Invitrogen), in 0.5% BSA in PBS with DAPI for 45 min at room temperature. Two washes with PBS were performed before imaging the cells on a Celigo Image Cytometer. Percent infection was quantified by dividing the number of NP-positive cells by the total number of cells. Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer's instructions, concurrent with immunostaining. Replication of A/WSN/33, A/Vietnam/1203/04, and A/Panama/99 were also tested using plaque assays and the IC50s for compound 2 were ˜2-fold less than in NP assays, indicating more inhibition of virus replication when assessing infectious particles.
For follow-up studies, compounds with the lowest AC50 in dose-response curves that showed retention of viral M mRNA in the nucleus were first selected by measuring nuclear to cytoplasmic ratios as in
The effect of compound 2 on the M mRNA nuclear export pathway was subsequently tested. It was previously shown that that M mRNA nuclear export is inhibited by knockdown of the mRNA export factor UAP56 (Mor et al., 2016, Nat Microbiol. 1(7): 16069; Wisskirchen et al., 2011, Journal of Virology 85(17): 8646-55; Read et al., 2010, The Journal of General Virology 91(Pt 5): 1290-301). This effect is also shown here with increasing concentrations of siRNAs that target UAP56 (
To further corroborate these data, the effect of a catalytically inactive mutant of UAP56 (E197A) was tested on nuclear export of viral M, HA, NS1, and poly(A) RNA. Cells stably expressing UAP56 (E197A) were generated as previously reported (Hondele et al., 2019, Nature 573(7772): 144-8). These cells were treated with control siRNA or with siRNA that targets the 3′UTR of UAP56—this siRNA depletes endogenous UAP56 and not UAP56 mutant. The efficiency of this siRNA is shown in
To quantitatively assess a potential impact of compound 2 on a subset of cellular RNAs and determine their identity, RNA-sequencing (RNA-seq) analysis was performed of purified poly(A) RNA obtained from whole cells, nuclear fractions, and cytoplasmic fractions either treated with DMSO (control) or with 2.5 μM of compound 2. As expected, RNAs that are known to be retained in nucleus, such as MALAT1, are primarilynuclear, and mRNAs that are distributed in the nucleus and cytoplasm, such as GAPDH mRNA, are shown in both compartments. A total of 19,799 unique RNAs were sequenced and the cutoff was 1.5-fold change to be considered differentially expressed in the presence of compound 2. It was shown that compound 2 altered the nuclear to cytoplasmic distribution of a small subset of cellular RNAs, including mRNAs and non-coding RNAs (
Since nuclear export of key viral mRNAs is blocked by compound 2 and given that these mRNAs encode critical proteins for the virus life cycle, it is expected that viral protein levels and replication would be altered by this compound. Indeed, there is a decrease in the levels of the viral M1 and M2 proteins as well as NA and HA proteins upon 2.5 μM compound treatment (FIG. 34A). Compound 2 was then tested for inhibition of virus replication and cytotoxicity. As expected, compound 2 inhibited replication of diverse influenza A virus strains at concentrations in which it did not significantly alter cell viability (
While antiviral treatments currently approved for clinical use target viral proteins directly, the presently disclosed compounds target cellular proteins without causing cytotoxicity. Because current antiviral compositions target viral proteins, such treatments have an increased probability of developing strains resistant to these antiviral compositions. In contrast, the presently disclosed compounds are effective against a variety of influenza viral strains as they target cellular proteins thus it is more difficult for antiviral resistant mutations to develop.
With the lack of robust and diverse medical interventions available, multiple antiviral strategies are needed to provide additional therapeutic options for influenza infections. One strategy is to identify viral-host interactions that can be targeted without compromising major host cellular functions. As the virus enters the host cell via endocytosis, the viral M2 ion channel on the viral membrane acidifies the interior of the virus particle. This enables viral uncoating and subsequent release of the viral genome into the host cytoplasm upon fusion of the viral and endosomal membranes. As the eight unique vRNPs enter the host cell nucleus, transcription initiates and 2 of the 8 viral mRNAs undergo alternative splicing. It is the alternative splicing event of the viral M1 mRNA into the viral M2 mRNA that generates the viral M2 protein that is key for viral entry. The M2 protein is also important for viral budding and inhibition of autophagy. M1 mRNA also encodes the M1 protein, which has key functions in viral intracellular trafficking and as a structural component of the infectious virions.
Based on knowledge of viral M mRNA trafficking through host nuclear speckles for splicing and nuclear export, a high-throughput screening strategy was designed that led to the identification of small molecules that interfered with specific steps of this pathway. The image-based chemical screening, which used single-molecule RNA-FISH, identified three classes of inhibitors that either decreased viral M mRNA levels (class 1), or blocked it in the nucleus (class 2), or both (class 3). Our primary HTS assay proved to be quite robust, as exemplified by an average Z′ value of 0.63 for the N/C ratio when comparing the DMSO (vehicle) control to a positive control, DRB. To ensure that all of the chemical space identified by the screen was sampled, the initial set of hits was clustered into chemical series for compounds that decreased the M mRNA fluorescence intensity (552 clusters, intensity reduced >25%) and for compounds that decreased the N/C ratio (˜1300 clusters, N/C ratio >25%). Cluster representatives were then selected from both groups as described above. Hit confirmation studies identified ˜600 compounds that fell into the three phenotypic classes described above. These compounds were subsequently reviewed for chemical attractiveness (e.g. absence of problematic substructures or PAINS, synthetic tractability, etc.). An inhibitor that preferentially prevented nuclear export of a subset of viral mRNAs (class 2) was further tested, resulting in accumulation in the nucleoplasm. Since this small molecule (and others like it identified by the screen) did not substantially alter bulk cellular mRNA levels or their intracellular distribution and were not cytotoxic at active concentrations, they may serve as leads for potential antiviral therapy. Therefore, these data revealed a window of opportunity to target a pathway that processes a subset of viral and cellular mRNAs. In addition, compound 2's differential nuclear export inhibition of viral mRNAs and cellular mRNAs demonstrates specific requirements within the mRNA export machinery for nuclear export and provides a tool to distinguish these pathways in future studies.
The differential effect of compound 2 on viral M mRNA nuclear export, phenocopying down-regulation of UAP56 activity, further corroborates its action on the UAP56-NXF1-mediated mRNA export pathway. This would be predicted based on the screening strategy presented here. UAP56 is known to recruit the mRNA export factor Aly/REF to the mRNA, which then binds the mRNA export receptor NXF1′NXT1. This interaction displaces UAP56 from the mRNA and NXF1′NXT1 then docks the mRNP to the nuclear pore complex for export into the cytoplasm. Prior to docking at the nuclear pore complex, the M mRNA is spliced at nuclear speckles and then exported to the nucleoplasm for translocation through the nuclear pore complex. UAP56 is localized at nuclear speckles and in the nucleoplasm and is required for exit of M mRNA from nuclear speckles to the nucleoplasm as previously shown (Mor et al., 2016, Nat Microbiol. 1(7): 16069). The localization and export function of UAP56 in the nucleoplasm and at nuclear speckles may involve different factors/adaptors. In contrast to M mRNA and a subset of cellular mRNAs whose splicing and/or export occur at nuclear speckles, most cellular mRNAs are spliced in the nucleoplasm prior to being exported from the nucleus. Compound 2 targets the viral M mRNA nuclear export without affecting its splicing at nuclear speckles. Therefore, it is likely that this small molecule is targeting a step between nuclear speckles and the nuclear pore complex, resulting in the accumulation of viral M mRNA throughout the nucleoplasm. Since bulk cellular mRNAs were not substantially affected by the compound at a concentration that it robustly inhibited M and HA mRNA nuclear export, it is possible that this compound is specifically targeting a step or location that affects a subset of cellular mRNAs. In fact, RNAseq analysis shows effect of compound 2 on nuclear export and total levels of a subset of cellular RNAs. This is consistent with the data in which partial depletion of UAP56 or expression of a UAP56 mutant in the catalytic domain in the presence of endogenous UAP56 preferentially blocked viral M and HA mRNA nuclear export without substantially altering NS1 mRNA or bulk cellular mRNAs. These differential effects by partially decreasing the levels of an mRNA export factor reveal a window of opportunity to therapeutically target the mRNA export machinery without inducing major cytotoxicity to the host cell.
Among the subset of cellular mRNAs whose total levels are up-regulated or down-regulated by compound 2 without changes in intracellular distribution, are a few mRNAs known to be regulated by the viral NS1 protein. In the category of up-regulated mRNAs are members of the Type-I interferon response system, including IFIT1 and IRF7 (Diamond, 2014, Cytokine Growth Factor Rev. 25(5): 543-50). IFN response is known to be suppressed by the NS1 protein therefore both IFIT1 and IRF7 mRNAs are up-regulated in cells infected with the influenza virus lacking NS1 protein. Regarding the down-regulated mRNAs, which were enriched in mRNAs that encode proteins involved in tyrosine metabolism, it is possible that the decrease in tyrosine metabolism inhibits virus replication. Tyrosine is a critical amino acid for viral proteins, such as tyrosine 132 phosphorylation of M1 protein which controls its nuclear import and virus replication. Additionally, virus replication is blocked by receptor tyrosine kinase inhibitors. Furthermore, 47 mRNAs in this down-regulated category are also regulated by NS1. Together, these data suggest that inhibition of influenza virus replication by compound 2 may be a combinatory effect of inhibition of viral mRNA export and induction of antiviral response which, at least in part, involves the Type-I interferon system.
Compound 2 is an alkylated mercaptobenzimidazole featuring an aminopyridine amide. No biological activities have been attributed to this compound previously. However, a structurally related series of N-aryl mercaptobenzimidazoles have been described as inhibitors of influenza viruses and myxoviruses. It was shown that the most potent compound of this series had no effect on M mRNA nuclear export (
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references.
This application is a continuation of International Patent Application Ser. No. PCT/US2021/070286 filed Mar. 18, 2021, which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/991,445 filed Mar. 18, 2020, the contents of which are hereby incorporated by reference in their entireties.
This invention was made with government support under Grant Number AI119304 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62991445 | Mar 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2021/070286 | Mar 2021 | US |
| Child | 17527907 | US |
