Patent Grant
11667678
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on Dec. 20, 2020, is named R635-US Sequence listing_ST25.txt and is 5 KB in size.
COVID-19 is an infectious respiratory illness caused by the virus strain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Common symptoms of COVID-19 are fever, cough, and shortness of breath and with a mortality rate of around 4-5%, it is more than 10 times lethal than the flu. While anyone is susceptible to COVID-19, the ones over 60 or with preexisting conditions, such as hypertension, obesity, asthma, or diabetes, are more vulnerable to severe symptoms (1, 2). Until now, no effective therapy is available to tackle this viral pandemic.
It is known that mutations in the viral genome are capable of altering the virus's pathogenic potential. Even an exchange of single amino acid may affect a virus's ability to escape the immune system and confuse the vaccine development progress against the virus. Consequently, since the beginning of SARS-CoV-2 in December 2019, variants have been emerging at a regular interval. It has also been discovered that SARS-CoV-2 variants are a significant health concern worldwide exhibiting increased transmissibility, cause more severe disease, exhibit evasive immune properties, impair neutralization by antibodies from vaccinated individuals or convalescence sera, and reinfection.
Although at least twelve different variants have been identified, there are several that are classified as “variants of concern” by the Center for Disease Control in the United States include B.1.1.7 (Alpha, also known as the “UK variant”), B.1.351 (Beta, also known as the “South Africa variant”), P.1 (Gamma also known as the “Brazil variant”), B.1.617.2 (Delta) and B.1.1.529 (Omicron). See Ramesh et al., “Emerging SARS-CoV-2 Variants: A Review of Its Mutations, Its Implications and Vaccine Efficacy,” Vaccines 2021: 9, 1195; Thakur et al., “OMICRON (B.1.1.529): A new SARS-CoV-2 variant of concern mounting worldwide fear,” J Med Virol. 2021; 1-4.
Most of the SARS-CoV-2 variants have mutations in spike glycoprotein, which is the primary target of different vaccines. See Ramesh et al. 2021. Therefore, it is seen that vaccines are not equally efficient against different SARS-CoV-2 variants (Al-Tawfiq et al., 2022). Accordingly, even after full vaccination with a booster, many people are being admitted in the ICU after infection with SARS-CoV-2 variants. Thus, it is of highest importance to study these variants and associated pathological properties.
Entry of SARS-CoV-2 into the host cells is probably the most important event in COVID-19 disease process. Angiotensin-converting enzyme 2 (ACE2), the main effector of the classical renin-angiotensin system, is a cell surface receptor that is predominant in lung, heart and kidney (3). Although the prototype function of ACE2 is to convert angiotensin II (AngII), a vasoconstrictor, to Ang1-7, a vasodilator, and thereby to play an important role in the pathophysiology of cardiovascular diseases (3, 4), recently, ACE2 came to renewed attention due to its requirement by COVID-19 for entering into host cells. It is found that COVID-19 binds to ACE2 via the S protein on its surface (2,5). During infection, the S protein is cleaved into S1 and S2 subunits and the S1 subunit encompasses the receptor-binding domain (RBD). Therefore, this subunit permits COVID-19 to directly attach to the peptidase domain of ACE2 (1,5).
Since ACE2 is a beneficial molecule, either inhibiting or knocking down of ACE2 is not a valid option. Since SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) for entering into host cells, to target COVID-19 from therapeutic angle, what is needed is an agent that inhibits the association between SARS-CoV-2 spike S1 and ACE-2. Further, what is needed are agents that inhibit the association between SARS-CoV-2 spike S1 and ACE-2 in the variants of concern that demonstrate increases infectivity and transmissibility.
Compositions are provided that include an isolated peptide that inhibits the association between SARS-CoV-2 spike S1 and Angiotensin Converting Enzyme-2 (ACE-2) without inhibiting the activity of ACE-2. In some embodiments, the compositions may comprise an isolated peptide consisting of 15 or fewer amino acids, wherein the peptide comprises at least 75% identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 9.
Methods for treating COVID-19 are provided. The methods include administering a first agent to a subject in need thereof, where the first agent comprising an isolated peptide that inhibits the association between SARS-CoV-2 spike S1 and Angiotensin Converting Enzyme-2 (ACE-2) without inhibiting the activity of ACE-2. In some embodiments, the methods include administering a first agent where the first agent comprises an isolated peptide consisting of 15 or fewer amino acids, wherein the peptide comprises at least 75% identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 9.
A method of making an in vivo model for COVID-19 is also provided. The method includes administering recombinant SARS-CoV-2 spike S1 protein nasally to an animal.
In addition, compositions are provided for inhibiting the association between SARS-CoV-2 spike S1 and Angiotensin Converting Enzyme-2 (ACE-2) without inhibiting the activity of ACE-2 in SARS-CoV-2 variants.
In other embodiments, methods are provided for treating infections with SARS-CoV-2 variants where the methods include administering a first agent to a subject in need thereof, where the first agent comprising an isolated peptide that inhibits the association between SARS-CoV-2 spike S1 and Angiotensin Converting Enzyme-2 (ACE-2) without inhibiting the activity of ACE-2.
The embodiments disclosed below are not intended to be exhaustive or to limit the scope of the disclosure to the precise form in the following description. Rather, the embodiments are chosen and described as examples so that others skilled in the art may utilize its teachings.
Compositions and methods for specific targeting of the binding between ACE2 and SARS-CoV-2 are provided. Recombinant peptides were designed corresponding to the ACE2-interacting domain of SARS-CoV-2 spike S1 (AIDS) that inhibited the binding between ACE2 and SARS-CoV-2 spike S1. Methods of treating COVID-19 using the peptides described herein are also provided. In some embodiments, the recombinant peptides specifically reduced spike S1-mediated induction of IL-6 and IL-1β in lung cells without modulating double-stranded RNA (poly IC)- and HIV-1 Tat-mediated expression of IL-6 and IL-1β.
An in vivo model of COVID-19 is also provided.
The term “amino acid” refers, in particular, to any one of the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) but also to non-proteinogenic and/or non-standard a-amino acids (such as, e.g., omithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, nordeucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group such as, e.g., cyclopentylaianine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cydohexylglycine, or phenylglycine) as well as β-amino acids (e.g., β-alanine), γ-amino acids (e.g., γ-aminobutyric acid, isoglutamine, or statine) and/or 6-amino acids as well as any other compound comprising at least one carboxylic acid group and at least one amino group. Unless defined otherwise, an “amino acid” preferably refers to an a-amino acid, more preferably to any one of the 20 standard proteinogenic a-amino acids (which can be present as the L-isomer or the D-isomer, and are preferably present as the L-isomer).
The terms “peptide” and “polypeptide”, are used herein interchangeably and refer to a polymer of two or more amino acids linked via amide bonds that are formed between an amino group of one amino acid and a carboxyl group of another amino acid. the term peptide or polypeptide, it is meant to include the peptide or polypeptide itself, as well as any physiologically acceptable salts thereof, or any chemically modification made thereto, which would be apparent or known to a person of ordinary skill in the art. The amino acids comprised in the peptide or polypeptide, which are also referred to as amino acid residues, may be selected from the 20 standard proteinogenic a-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Iie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) but also from non-proteinogenic and/or non-standard a-amino acids (such as, e.g., omithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, a-methylalanine (i.e., 2-aminoisobutyric acid), norvaline, norleucine, terleucine (tert-leucine), labionin, or an alanine or glycine that is substituted at the side chain with a cyclic group such as, e.g., cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienylalanine, cyclohexylglycine, or phenylglycine) as well as β-amino acids (e.g., β-alanine), γ-amino acids (e.g., γ-aminobutyric acid, isogiutamine, or statine) and δ-amino acids. Preferably, the amino acid residues comprised in the peptide or polypeptide are selected from a-amino acids, more preferably from the 20 standard proteinogenic a-amino acids (which can be present as the L-isomer or the D-isomer, and are preferably all present as the L-isomer). The peptide or polypeptide may be unmodified or may be modified, e.g., at its N-terminus, at its C-terminus and/or at a functional group in the side chain of any of its amino acid residues (particularly at the side chain functional group of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and/or Arg residues). Such modifications may include, e.g., the attachment of any of the protecting groups described for the corresponding functional groups in: Wuts P G & Greene T W, Greene's protective groups in organic synthesis, John Wiley & Sons, 2006. Such modifications may also include the covalent attachment of one or more polyethylene glycol (PEG) chains (forming a PEGylated peptide or polypeptide), the glycosylation and/or the acylation with one or more fatty acids (e.g., one or more C8 30 alkanoic or alkenoic acids; forming a fatty acid acylated peptide or polypeptide). Moreover, such modified peptide or proteins may also include peptidomimetics, provided that they contain at least two amino acids that are linked via an amide bond (formed between an amino group of one amino acid and a carboxyl group of another amino acid). The amino acid residues comprised in the peptide or polypeptide may, e.g., be present as a linear molecular chain (forming a linear peptide or protein) or may form one or more rings (corresponding to a cyclic peptide or polypeptide). The peptide or polypeptide may also form oligomers consisting of two or more identical or different molecules.
The term “identity” refers to the overall relatedness between polymeric molecules, e.g., between peptides or polypeptides. Methods for the calculation of a percent identity as between two provided polypeptide sequences are known. Calculation of the percent identity of two polypeptide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences may be disregarded for comparison purposes). The amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, such as BLAST (basic local alignment search tool). In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).
To calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of an algorithm available for comparison of amino acid or nucleic acid sequences, comprising those available in commercial computer programs is BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying similar sequences, the programs mentioned above generally provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be substantially similar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of their corresponding residues are similar and/or identical over a relevant stretch of residues (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments, the relevant stretch is a complete sequence.
The term “subject” or “patient” as used herein, refers to a mammal, in some aspects a human.
A “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent, e.g., a peptide, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The therapeutic agent may inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent a disorder, and/or any one of the symptoms of the disorder. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
“Treating”, “treat”, or “treatment” within the context of the instant disclosure, means an alleviation of symptoms associated with a disorder or disease, or halt of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder. For example, within the context of this disclosure, successful treatment may include an alleviation of symptoms related to COVID-19. The treatment may include administering an effective amount of a peptide to the subject that results in an alleviation of symptoms associated with a disorder or disease, or halt of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder.
In some embodiments, practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, immunology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, (Current Edition); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (Current Edition)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (Current Edition) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)). DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.)
Recombinant Peptides
Recombinant peptides were designed corresponding to the ACE2-interacting domain of SARS-CoV-2 spike S1 (AIDS) and the Spike S1-Interacting Domain of ACE2 Receptor (SPIDAR) that inhibited the binding between ACE2 and SARS-CoV-2 spike S1. The recombinant peptides used herein may be constructed solely of natural amino acids. Alternatively, the recombinant peptides may include non-natural amino acids including, but not limited to, modified amino acids. Modified amino acids include natural amino acids which have been chemically modified to include a group or groups not naturally present on the amino acid. The recombinant peptides may additionally include D-amino acids. Still further, the recombinant peptides may include amino acid analogs.
Recombinant peptides that inhibit the binding between ACE2 and SARS-CoV-2 spike S1 may include a core sequence of NGVGY (SEQ ID NO: 3) and peptides having at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 75-80%, 80-85%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) thereto. In some embodiments, the recombinant peptide may have at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 75-80%, 80-85%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identity to a core sequence TNGVGY (SEQ ID NO: 4). In some embodiments, the recombinant peptide may have at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 75-80%, 80-85%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identity to QSYGFQPTNGVGY (SEQ ID NO: 5). In some embodiments, the core sequence of SEQ ID NO: 2 targets amino acids 501-505 of the SARS-CoV-2 spike S1 protein. In yet other embodiments, the recombinant peptides may have at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 75-80%, 80-85%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identity to a core sequence PLQSYG (SEQ ID NO: 6). In yet other embodiments, the recombinant peptides may have at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 75-80%, 80-85%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identity to a core EDLFYQS (SEQ ID NO: 7). In yet other embodiments, the recombinant peptides may have at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 75-80%, 80-85%, 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) identity to a core EDLFYQ (SEQ ID NO: 9). In some embodiments, one, two, three or more recombinant peptides may be used together. By way of non-limiting example, the recombinant peptides of SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5 may be combined with SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 9 and delivered to a subject. In some embodiments, the recombinant peptides disclosed herein may be in a composition comprising an isolated peptide consisting of 15 or fewer amino acids.
In some embodiments, the recombinant peptides disclosed herein may be in a composition comprising an isolated peptide consisting of 15 or fewer amino acids and include a core sequence of 5, 6 or 7 amino acids plus up to 10, 9 or 8 additional amino acids, respectively, added to the N-terminus or C-terminus or both the N-terminus and the C-terminus of the core sequence. In some embodiments, the recombinant peptides disclosed herein may be in a composition comprising an isolated peptide consisting of 15 or fewer amino acids and include a core sequence of 5, 6 or 7 amino acids plus up to 10, 9 or 8 additional amino acids, respectively, added to the N-terminus or C-terminus or both the N-terminus and the C-terminus of the core sequence (shown in bold) in consecutive order from the core sequence based on PLQSYGFQPTNGVGYQPYRVVVLSF (SEQ ID NO: 11) or FLDKFNHEAEDLFYQSSLASWNYN (SEQ ID NO: 12) and extending in either direction for a total of 15 or fewer amino acids. In some embodiments, the recombinant peptides disclosed herein may be in a composition comprising an isolated peptide consisting of 15 or fewer amino acids and include a core sequence of 5, 6 or 7 amino acids plus fewer than 10, 9 or 8 additional amino acids, respectively, added to the N-terminus or the C-terminus or both the N-terminus and the C-terminus of the core sequence. In some embodiments, the recombinant peptide consists essentially of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 9 or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 9 having one or two amino acid variations, in the core sequence or added to the N-terminus or the C-terminus or both the N-terminus and the C-terminus of the core sequence.
In some embodiments, the composition may include an addition peptide to dear SARS-CoV-2 from central nervous system (CNS). The additional peptide may facilitate penetrance of the isolated peptide that inhibits the binding between ACE2 and SARS-CoV-2 spike S1 into the CNS. In some embodiments the additional peptide may be from antennapedia homeodomain. In some embodiments, the cell penetrating peptide may be DRQIKIWFQNRRMKWKK (SEQ ID NO: 13). The cell penetrating peptide may be added to the N-terminus or the C-terminus of any of the peptides described above that inhibit the binding between ACE2 and SARS-CoV-2 spike S1.
By way of non-limiting example, the AIDS peptide for clearing SARS-CoV-2 from the brain and spinal cord may be: DRQIKIWFQNRRMKWKKTNGVGY (SEQ ID NO: 14) and the SPIDAR peptide for clearing SARS-CoV-2 from the brain and spinal cord may be: DRQIKIWFQNRRMKWKKEDLFYQ (SEQ ID NO: 15). In some embodiments, any of the AIDS peptide family members may be coupled to the cell penetrating peptide. In some embodiments, any of the SPIDAR peptide family members may be coupled to the cell penetrating peptide.
Pharmaceutical Compositions
The peptides described herein may be used alone or in compositions together with a pharmaceutically acceptable carrier or excipient. Pharmaceutical compositions of the disclosed herein may comprise a therapeutically effective amount of one or more peptides, together with one or more pharmaceutically acceptable carriers. In some embodiments, the peptide is used alone with a pharmaceutically acceptable carrier or excipient such that no other active agent is administered with the peptide.
As used herein, the term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringers solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Other suitable pharmaceutically acceptable excipients are described in “Remington's Pharmaceutical Sciences,” Mack Pub. Co., New Jersey, 1991, incorporated herein by reference.
The peptides described herein may be administered to humans and animals in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions for use in the present disclosure can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules or lipid particles, lyophilized powders, or other forms known in the art.
Peptides as used herein may be formulated for delivery as a liquid, aerosol or inhalable dry powder. Peptides may be delivered nasally or by inhalation. Liquid aerosol formulations may be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the peptide, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the peptide is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
The peptides can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the peptide may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
Dosage forms for topical or transdermal administration of a peptide include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of this disclosure.
The ointments, pastes, creams and gels may contain, in addition to a peptide, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
The peptides may also be formulated for use as topical powders and sprays that can contain, in addition to the peptide, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches may also be used for providing controlled delivery of a peptide to the body. Such dosage forms can be made by dissolving or dispensing the peptide in the proper medium. Absorption enhancers can also be used to increase the flux of the peptide across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the peptide in a polymer matrix or gel. The peptide can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a peptide, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott (ed.), “Methods in Cell Biology,” Volume XIV, Academic Press, New York, 1976, p. 33 et seq.
Aerosolized formulations of the peptides described herein may be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of an aerosol particles having with a mass medium average diameter predominantly between 1 to 5 μm. Further, the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosolizable volume able to deliver effective dose of the peptide to the site of treatment. Additionally, the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.
Aerosolization devices suitable for administration of aerosol formulations of the peptide include, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the peptide into aerosol particle size predominantly in the size range from 1-5 μm. Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are within 1-5 μm range. A jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. A variety of suitable devices are available, including, for example, AERONEB and AERODOSE vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, Calif.), SIDESTREAM nebulizers (Medic-Aid Ltd., West Sussex, England), PARI LC and PARI LC STAR jet nebulizers (Pan Respiratory Equipment, Inc., Richmond, Va.), and AEROSONIC (DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden, Germany) and ULTRAAIRE (Omron Healthcare, Inc., Vernon Hills, Ill.) ultrasonic nebulizers.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-propanediol or 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringers solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.
A peptide described herein can be administered alone or in combination with other agents, for a possible combination therapy being staggered or given independently of one another. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after the initial treatment, or even preventive therapy, for example in patients at risk.
Effective amounts of the peptide generally include any amount sufficient to detectably an inhibition or alleviation of symptoms. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific peptide employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
If the peptide is administered in combination with another compound, the term “amount that is effective” is understood to mean that amount of peptide in combination with the additional compound to achieve the desired effect. In other words, a suitable combination therapy according to the current disclosure encompasses an amount of the peptide and an amount of the additional compound, either of which when given alone at that particular dose would not constitute an effective amount, but administered in combination would be an “amount that is effective”.
It will be understood, however, that the total daily usage of the peptide and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific peptide employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific peptide employed; the duration of the treatment; drugs used in combination or coincidental with the specific peptide employed; and like factors well known in the medical arts.
The dose of a peptide to be administered to warm-blooded animals, for example humans of approximately 70 kg body weight, is preferably from approximately 3 mg to approximately 5 g, more preferably from approximately 10 mg to approximately 1.5 g, most preferably from about 100 mg to about 1000 mg per person per day, divided preferably into 1 to 3 single doses which may, for example, be of the same size. Usually, children receive half of the adult dose.
In Vivo Model of SARS-CoV-2
An in vivo model for COVID-19 is included herein. The in vivo model is a virus free model that develops one or more symptoms of COVID-19 including but not limited to fever, augmented lung inflammation, increased infiltration of neutrophils into the lungs, decreased heart function, and impaired locomotor activities relative to a control animal. The in vivo model is prepared by administering recombinant SARS-CoV-2 spike S1 protein nasally to an animal. By way of non-limiting example, the recombinant SARS-CoV-2 spike S1 may be SEQ ID NO; 1 although other sequence variants of SARS-CoV-2 spike S1 may also be used. In some embodiments, the recombinant SARS-CoV-2 spike S1 may be delivered nasally to one or both nostrils of the animal. In some embodiments, the recombinant SARS-CoV-2 spike S1 may be delivered by inhalation. In some embodiments, the recombinant SARS-CoV-2 spike S1 protein is delivered in a saline solution.
Results
Designing of a peptide corresponding to the ACE2-interacting domain of SARS-CoV-2 (AIDS): Since there is no specific treatment for COVID-19, from the therapeutic angle, we decided to target the interaction between SARS-CoV-2 and its receptor ACE2. The receptor-binding domain (RBD) of SARS-CoV-2 spike S1 is involved in the interaction with ACE26. Therefore, we applied rigid-body protein-protein interaction tool to model the interaction between RBD of spike protein S1 subunit and ACE2. As evident from our in silico modeling analysis, the docked pose of ACE2 and S1 RBD complex revealed a strong H-bond between Asn501 of spike S1 and Lys353 of ACE2. In addition, Tyr505 of spike S1 exhibited a hydrophobic interaction with Gln42 and the side chain of Lys353 of ACE2 (
Wild type (wt) AIDS: 500TNGVGY505 (SEQ ID NO: 4)
Mutated (m) AIDS: 500TGGVGD505 (SEQ ID NO: 8)
Positions of mutations are underlined. Next, to examine whether wtAIDS peptide inhibits the binding of ACE2 with SARS-CoV-2 spike S1, we employed chemiluminescence-based ACE2:SARS-CoV-2 spike S1 binding using an assay kit (catalog #79936; BPS Bioscience). As evident from
AIDS peptide inhibits lung cell inflammation induced by SARS-CoV-2 spike S1, but not double-stranded RNA (poly IC), HIV-1 Tat, and bacterial flagellin: Pulmonary inflammation ultimately leading to acute lung injury is becoming a hallmark of COVID-19 patients visiting ICU′. In addition to COVID-19, pulmonary complications are also evident in different bacterial and viral infections8,9. Therefore, we investigated if AIDS peptide was capable of suppressing the expression of proinflammatory molecules in human A549 lung cells induced by different stimuli. A549 cells pretreated with different concentrations of wtAIDS and mAIDS peptides for 15 min were stimulated with recombinant SARS-CoV-2 spike S1, poly IC, HIV-1 Tat, and bacterial flagellin. At first, we examined whether recombinant SARS-CoV-2 spike S1 was capable of inducing proinflammatory cytokines in A549 lung cells. Dose-dependent analysis showed that SARS-CoV-2 spike S1 was very potent in inducing proinflammatory cytokines and that spike S1 even at a dose of 0.2 ng/ml significantly induced the mRNA expression of IL-6 and IL-1β in lung cells with maximum induction at 1 ng/ml (
Similarly, poly IC (
Intranasal administration of SARS-CoV-2 spike S1 causes fever and lung inflammation: Protection by wtAIDS peptide: One of the most common symptoms of COVID-19 is fever2,10. Interestingly, daily intranasal administration of SARS-CoV-2 spike S1 at a very low dose (
The wtAIDS peptide recovers heart functions and improves locomotor activities in SARS-CoV-2 spike S1-intoxicated mice: Many COVID-19 patients in the ICU develop cardiac arrhythmias13. Therefore, we examined if these cardiac features of COVID-19 could be modeled in SARS-CoV-2 spike S1-intoxicated mice. Different cardiac parameters are schematically presented in
Next, to examine whether spike S1 intoxication also caused functional deficits, we monitored locomotor and open-field activities. Spike S1 insult decreased overall locomotor activities as evident by heat map (
Discussion
Until now, almost 900,000 people died throughout the world due to COVID-19. Therefore, untangling the mechanism of the disease process of COVID-19 and designing an effective therapeutic approach to slow down the disease and stop the death are of paramount importance. ACE2 being the main player of the classical renin-angiotensin pathway plays an important role in vascular diseases3. Since SARS-CoV-2 binds ACE2 and causes its internalization to enter into human cells, COVID-19 is particularly deleterious to patients with underlying cardiovascular issues. Although ACE2 inhibitors are available14 and such inhibitors may stop the entry of SARS-CoV-2 into human cells, it is not possible to inhibit this beneficial molecule. Therefore, through structural analysis of the interaction between SARS-CoV-2 and ACE2, we have designed a small hexapeptide corresponding to the ACE2-interacting domain of SARS-CoV-2 (AIDS). Since the spike S1 of SARS-CoV-2 interacts with ACE2, wtAIDS peptide unsettled the association between ACE2 and SARS-CoV-2 spike S1.
Although it has been shown that a subgroup of patients with severe COVID-19 symptoms suffer from cytokine storm15, underlying mechanism was not known. Marked induction of proinflammatory cytokines (IL-6 and IL-1β) in human A549 lung cells by SARS-CoV-2 spike S1 even at a very low dose suggests that this spike subunit may contribute to cytokine storm in COVID-19 patients. Consistent to the inhibition of association between ACE2 and SARS-CoV-2 spike S1, wtAIDS peptide inhibited the expression of IL-6 and IL-1β in SARS-CoV-2 spike S1-intoxicated A549 lung cells. In contrast, wtAIDS peptide did not inhibit the expression of proinflammatory cytokines induced by poly IC (viral double-stranded RNA mimic), Tat (transactivator of HIV-1 transcription) and flagellin (a component of bacterial infection), indicating the selective nature of wtAIDS peptide. Most importantly, the beauty of our finding is that wtAIDS peptide corresponds to peptide sequence of SARS-CoV-2. Therefore, it will only inhibit the binding of SARS-CoV-2 with ACE2 without affecting basal level and beneficial functions of ACE2. Moreover, it will function only in the presence of SARS-CoV-2.
Developing a small animal model system is an important step in understanding mechanisms associated to deadly cardiovascular and pulmonary issues of COVID-19 and evaluating effective drugs for this global pandemic. While handling live SARS-CoV-2 has many biosafety requirements, here, we describe the development of a very simple and live virus-free model of mimicking important cardiac and respiratory symptoms of COVID-19 in mice. SARS-CoV-2 spike S1 containing the RBD sequence is located at the N-terminus of the spike protein. Since most of the neutralizing epitopes are located within the spike S1, this spike subunit has become an important candidate for vaccine development. Interestingly, here, we demonstrated that intranasal administration of the same spike S1 was capable of inducing many key features of COVID-19 (fever, lung inflammation, lung neutrophil infiltration, increase in serum IL-6, and arrhythmias) in normal mice. Therefore, nasally SARS-CoV-2 spike S1-intoxicated mice could be used as a virus-free mouse model for testing different therapeutic options against COVID-19.
Until now, there is no effective therapy and also no approved vaccines for COVID-19. Although hydroxychloroquine showed some promise in the beginning, several clinical trials have ruled out the use of hydroxychloroquine in COVID-1910. Only, Remdesivir has been approved for emergency use in COVID-1916. Although s.c. injection of some ICU patients with IFN β-1 b led to decrease in mortality rate17, further randomized clinical trials with large sample size are needed for the exact estimation of survival benefit by this multiple sclerosis drug. Reduction of fever, protection of lungs, normalization of heart functions, and improvement in locomotor activities in SARS-CoV-2 spike S1-intoxicated mice by intranasal treatment with wtAIDS peptide suggest that selective targeting of SARS-CoV-2:ACE2 contact by wtAIDS peptide may be beneficial for COVID-19. The wtAIDS peptide should target only the SARS-CoV-2-dependent ACE2 pathway without inhibiting normal physiological functions of ACE2. Accordingly, in a group of control mice, we also did not notice any drug-related side effect (e.g. hair loss, appetite loss, weight loss, untoward infection and irritation, etc.) upon treatment with intranasal wtAIDS peptide. In summary, our preclinical studies have identified intranasal AIDS peptide as a primary or adjunct therapeutic option for COVID-19 patients.
Materials and Methods
Reagents
Human A549 lung carcinoma cell line (cat #CCL-185) and F-12K medium (cat #30-2004) were purchased from ATCC. Hank's balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic were purchased from Mediatech (Washington, D.C.). Fetal bovine serum (FBS) was obtained from Atlas Biologicals. ACE2:SARS-CoV-2 Spike Inhibitor Screening Assay Kit (Cat #79936) was purchased from BPS Bioscience. Recombinant COVID-19 Spike protein S1 was purchased from MyBioSource (Cat #MBS553722) and Abeomics (Cat #MBS553722). Anti-SARS-CoV-2 Spike S1 antibody (Cat #A3000-50) was bought from BioVision. Human IL-1β ELISA and IL-6 ELISA kits were bought from ThermoFisher.
Animals and Intranasal Delivery of AIDS Peptides
Mice were maintained and experiments conducted in accordance with National Institute of Health guidelines and were approved by the Rush University Medical Center IACUC. C57/BL6 mice (6-8 week old; Envigo) of both sexes were treated intranasally with wtAIDS or mAIDS peptides (100 ng/mouse/d) for 7 d. Briefly, AIDS peptides were dissolved in 2 μl normal saline, mice were hold in supine position and 1 μl volume was delivered into each nostril using a pipetman.
Typically, any animal experiment is justified with 99% confidence interval that generates p=0.99 and (1-p)=(1-0.99)=0.01; £ is the margin of error=0.05. Based on these value, the resultant sample size is:
Therefore, six mice (n=6) were used in each group.
Intoxication of C57/BL6 mice with recombinant SARS-CoV-2 spike S1
C57/BL6 mice (6-8 week old; Envigo) of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) intranasally. Briefly, recombinant spike S1 was dissolved in 2 μl normal saline, mice were hold in supine position and 1 μl volume was delivered into each nostril using a pipetman. Control mice received only 2 μl saline.
Non-Invasive ECG Recording
Prior to ECG recording, mice were acclimatized to the ECG pulse transducer pad (AD instruments TN 012/ST, USA) and the experimental housing conditions. ECG pulse transducer pad was placed around the heart of each animal and ECG recording was carried out for 120 s. For ECG analysis, electrocardiography data were exported from the Labchart pro, version 8.0 (Power Lab 4/35 model) as raw data format and the digital signal processing was performed using this software. The recording was conducted for 120 s and the ECG signals were recorded at a sampling range of 20 mV with 4 beats/s sampling rate as recommended in the software for mouse ECG analysis. The ECG tracing was visually reviewed for identifying possible arrythmias or other anomalous complexes. Prior to analyze the data, in the Labchart pro software ECG setting data source was kept for ECG channel selected as the channel 1 out of the multiple channels and selected for whole channel specific for mouse species. In the detection setting, typical QRS width was reserved at 10 ms and R wave kept at least 60 ms apart with the alignment maintained at QRS maximum. In the analysis portion, pre-baseline was kept at 10 ms with maximum at 50 ms. We selected for rodent waves and measured ST segment height at 10 ms height. All the ECG parameters including PQ interval, QRS interval, QT interval, T duration, JT interval as well as P Amplitude, Q amplitude, R Amplitude, S amplitude, T amplitude were calculated. The recording and analysis settings were kept same for all the experimental mice included in this study.
Monitoring Lung Infiltration and Pathology
After treatment, animals were anesthetized with ketamine/xylazine injectable followed by transcardial perfusion18. The lungs were collected and processed for histological studies. Hematoxylin-eosin (HE) (SigmaAldrich, St Louis, Mo.) staining was performed from 4 μm thick paraffin embedded sections and used for studying the general lung tissue morphology. Number of epithelial cells, and the number infiltrated neutrophils in alveolar spaces and interstitial space were analyzed by ImageJ. At least ten 40× fields from each group were chosen for the counting of the epithelial and infiltrated neutrophils.
In Silico Structural Analysis
In silico structural analysis was performed as described earlier19,20. Briefly, by utilizing the protein preparation tools from the Schrodinger, Inc. platform, we evaluated the quality of the crystal structure of human ACE2 and SARS-CoV-2 spike S1. After assessing the quality of the crystal structure, hydrogens were added to the hydrogen bond orientation, charges were added in the Optimized Potential for Liquid Simulations (OPLS3) force field followed by adding missing atoms and side chains of the different residues of both the proteins. Finally, the complex structure was subjected to energy minimization in OPLS3 force field to make it torsion free. After the protein predation, we extracted out the spike protein from the ACE2 and then applied the dynamic hydrogen bonding module in order to find potential hydrogen bonds between the two structures. After assessing the H-bonds, we also evaluated other interactions such as hydrophobic interactions between the two structures as shown in
ACE2:SARS-CoV-2 Spike Binding Assay
The effect of wtAIDS and mAIDS peptides on the binding of ACE2 and SARS-CoV-2 spike was examined using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, Calif.) according to manufacturer's instructions. Briefly, 96-well nickel-treated plate provided by the manufacturer was coated with ACE2 solution. After washing with immuno buffer and incubation with blocking buffer, different concentrations of AIDS peptides were added to each well followed by addition of SARS-CoV-2 Spike (RBD)-Fc. After washing and incubation with blocking buffer, plates were treated with anti-mouse Fc-HRP followed by addition of an HRP substrate. Resultant chemiluminescence was measured using Perkin Elmer multimode microplate reader, Victor X5.
Semi-Quantitative RT-PCR Analysis
Total RNA was isolated from A549 lung cells and mouse lungs using RNAeasy Mini kit (Qiagen, Germantown, Md.) and Ultraspec-II RNA reagent (Biotecx Laboratories, Inc., Houston, Tex.), respectively. To remove any contaminating genomic DNA, total RNA was digested with DNase. RT-PCR was carried out as described earlier21,22 using a RT-PCR kit (Clontech, Mountain View, Calif.). Amplified products were electrophoresed on a 1.8% agarose gels and visualized by ethidium bromide staining. Message for the GAPDH gene was used to ascertain that an equivalent amount of cDNA was synthesized from different samples.
Real-Time PCR Analysis
DNase-digested RNA was analyzed by real-time PCR in the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, Calif.) as described earlier21,22.
ELISA for IL-6
IL-6 ELISA was performed in lung homogenates as described earlier23 using an assay kit (eBioscience) according to manufacturer's instruction.
Statistical Analysis
Statistical analyses were perfumed using Graphpad prism 8.0 (GraphPad Software, Inc., La Jolla, Calif.). Mouse behavioral measures were examined by an independent one-way ANOVA using SPSS. Homogeneity of variance between test groups was examined using Levene's test. Post-hoc analyses were conducted using Tukey's tests. Other data were expressed as means±SD of three independent experiments. Statistical differences between means were calculated by the Student's t-test (two-tailed). A p-value of less than 0.05 (p<0.05) was considered statistically significant.
Angiotensin-converting enzyme 2 (ACE2) on host cells serves as the receptor for SARS-CoV-2, and therefore, for targeting COVID-19 from therapeutic angle, we designed a hexapeptide corresponding to the Spike S1-Interacting Domain of ACE2 Receptor (SPIDAR) (
37EDLFYQ42
37EKLFYG42
Positions of mutations are underlined.
Intranasal treatment with wtSPIDAR as described above, but not mSPIDAR, decreased the level of proinflammatory molecules in lungs and serum of a mouse model of COVID-19 (
Studies will be conducted to determine the effects of different peptides that inhibited the binding between ACE2 and SARS-CoV-2 spike S1 using the protocols described in Example 1. By way of non-limiting example, studies will be conducted using peptides having at least 80% identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
Effect of wtSPIDAR (SEQ ID NO: 9) on IL-6 Promoter-Driven Luciferase Activity in Human A549 Lung Cells Stimulated with Spike S1 of Different Variants of SARS-CoV-2
The cytokine storm is associated with a severe symptom of acute respiratory distress syndrome (ARDS) found in some COVID-19 patients. See Desai et al., “Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection,” Nature Comm. (2020) 11:6319, https://doi.org/10.1038/s41467-020-20139-7. Recently it has been observed that SARS-CoV-2 spike S1 is capable of inducing the expression of different proinflammatory cytokines in human A549 lung cells and that wtSPIDAR markedly suppresses SARS-CoV-2 spike S1-induced expression of proinflammatory cytokines. See herein and Paidi et al., “ACE-2-interacting Domain of SARS-CoV-2 (AIDS) Peptide Suppresses Inflammation to Reduce Fever and Protect Lungs and Heart in Mice: Implications for COVID-19 Therapy,” J Neuroimmune Pharmac (2021) 16:59-702021; Paidi et al., “Selective Inhibition of the Interaction between SARS-CoV-2 Spike S1 and ACE2 by SPIDAR Peptide Induces Anti-Inflammatory Therapeutic Responses,” J. Immunol. (2021) doi:10.4049/jimmunol.2100144. Since many variants (e.g. Delta and Omicron) evade the protection provided by COVID-19 vaccines, the current studies were undertaken to delineate whether wtSPIDAR exhibited any efficacy against delta variant SARS-CoV-2 spike S1, Alpha (N501Y or UK) variant SARS-CoV-2 spike S1, Beta (E484K or South Africa) variant SARS-CoV-2 spike S1, and Omicron variant SARS-CoV-2 spike S1 in human A549 lung cells.
In addition, the inventors investigated whether naturally available molecules would be useful in this model as well. For example, prenol is a fruit alcohol and it is present in different fruits and is known to have medicinal properties. Similarly, 4-hydroxybenzoate (4HB) is present in several different plants including medical mushrooms and coconut. Accordingly, it is expected that these molecules should be much less toxic as compared to synthetic drugs. Therefore, the efficacy of these molecules was also tested in dissociating the interaction between SARS-CoV-2 spike S1 and ACE2 and hence viral entry into human cells.
U.K. variant (Alpha or N501Y) SARS-CoV-2 spike S1 and South Africa variant (Beta or E484K) SARS-CoV-2 spike S1 were obtained from BioVision Inc., Milpitas, Calif. Delta variant SARS-CoV-2 spike S1 was obtained from Abbexa, Cambridge, UK.
For comparison, cells were also treated with 4-hydroxy benzoate (4HB) and prenol because in other experiments, we have noticed efficacy of these nontoxic molecules against SARS-CoV-2 spike S1 toxicity.
A549 cells cells plated at 70-80% confluence in 12-well plates were transfected with 0.25 μg pIL-6 promoter-Luc using Lipofectamine-Plus (ThermoFisher). After 24 h of transfection, cells were treated with different doses of 4HB, prenol and wtSPIDAR for 15 min followed by stimulation with 10 ng/ml of Delta variant, UK variant and South Africa variant SARS-CoV-2 spike S1 under serum-free condition. After 4 h of stimulation, firefly luciferase activities were measured in total cell extracts in a TD-20/20 Luminometer (Turner Designs) using the Luciferase Assay System kit (Promega).
As expected, Delta variant SARS-CoV-2 spike S1, N501Y SARS-CoV-2 spike S1 and E484K SARS-CoV-2 spike S1 induced IL-6 promoter-driven luciferase activity in A549 cells. However, significant inhibition of reporter activity was found by treatment with wtSPIDAR, 4HB and prenol.
Thus, all of the results indicate the ability of wtSPIDAR alone or in combination with 4HB and prenol can be useful treatments for infections with different variants of SARS-CoV-2.
The above Figures and disclosure are intended to be illustrative and not exhaustive. This description will suggest many variations and alternatives to one of ordinary skill in the art. All such variations and alternatives are intended to be encompassed within the scope of the attached claims. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the attached claims.
This application is a continuation-in-part application of U.S. application Ser. No. 17/133,035, filed Dec. 23, 2020, which claims the benefit of the filing date of U.S. Provisional Patent Application No. 63/078,547, filed Sep. 15, 2020 and U.S. Provisional Patent Application No. 62/704,091, filed Apr. 21, 2020, the contents of which are hereby incorporated by reference.
This invention was made with Government support of Grant Numbers AG050431, AT010980 and NS108025 awarded by the National Institutes of Health and Grant Number 11K6 BX004982 awarded by the United States Department of Veterans Affairs. The Federal Government has certain rights in this invention.
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| 20220363722 A1 | Nov 2022 | US |
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| 63078547 | Sep 2020 | US | |
| 62704091 | Apr 2019 | US |
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| Parent | 17133035 | Dec 2020 | US |
| Child | 17699792 | US |
