U.S. patent application number 17/199342 was filed with the patent office on 2021-10-28 for mers-cov inhibitor peptides.
The applicant listed for this patent is KING FAISAL UNIVERSITY. Invention is credited to MOHAMMED AL-NAZAWI, ABDULLA YOUSEF AL-TAHER, MAHMOUD KANDEEL ELSAYED, HYUNG-JOO KWON.
Application Number | 20210332086 17/199342 |
Document ID | / |
Family ID | 1000005449605 |
Filed Date | 2021-10-28 |
United States Patent
Application |
20210332086 |
Kind Code |
A1 |
ELSAYED; MAHMOUD KANDEEL ;
et al. |
October 28, 2021 |
MERS-COV INHIBITOR PEPTIDES
Abstract
The MERS-CoV inhibitor peptides include a set of peptides
designed by modification or mutation of a wild type MERS-CoV fusion
protein. The MERS-CoV inhibitor peptides are capable of inhibition
of MERS-CoV membrane fusion, and thereby may prevent or slow the
spread of MERS-CoV infections. Thus, the MERS-CoV inhibitor
peptides may be used in pharmaceuticals to prevent and/or treat
MERS-CoV infection. The pharmaceuticals may be formulated to
comprise at least one of the MERS-CoV inhibitor peptides and a
carrier, or they may include one or more expression systems capable
of promoting cellular expression of one or more MERS-CoV inhibitor
peptides. The MERS-CoV inhibitor peptides may also be used as
reagents for MERS-CoV inhibition assays as a standard or reference
inhibitors.
Inventors: |
ELSAYED; MAHMOUD KANDEEL;
(AL-AHSA, SA) ; AL-TAHER; ABDULLA YOUSEF;
(AL-AHSA, SA) ; KWON; HYUNG-JOO; (AL-AHSA, SA)
; AL-NAZAWI; MOHAMMED; (AL-AHSA, SA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KING FAISAL UNIVERSITY |
AL-AHSA |
|
SA |
|
|
Family ID: |
1000005449605 |
Appl. No.: |
17/199342 |
Filed: |
March 11, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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16857136 |
Apr 23, 2020 |
10975126 |
|
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17199342 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61K 38/00 20130101; C07K 14/005 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005 |
Claims
1-5. (canceled)
6. A method for conducting a MERS CoV inhibition assay comprising
using a MERS CoV inhibitor as a standard or as a reference
inhibitor, wherein the MERS CoV inhibitor comprises a peptide
having the amino acid sequence selected from the group consisting
of SEQ ID NOs: 4-6 and a combination thereof.
7. The method of claim 6, wherein the assay is a cell-cell fusion
assay.
8. The method of claim 6, wherein the the assays is a MERS CoV
plaque formation assay.
9. A pharmaceutical composition consisting of a MERS CoV inhibitor
and a pharmaceutically acceptable carrier, wherein the MERS CoV
inhibitor comprises a peptide having the amino acid sequence
selected from the group consisting of SEQ ID NOs: 4-6 and a
combination thereof.
10-14. (canceled)
15. A method of preventing MERS CoV infection of a cell comprising:
administering a composition comprising at least one peptide having
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 4-6, and a combination thereof.
16.-19. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of application Ser. No.
16/857,136, filed Apr. 23, 2020, pending, the priority of which is
claimed.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED IN COMPUTER
READABLE FORM
[0002] The Applicants hereby incorporate by reference the sequence
listing contained in the ASCII text filed titled
32087_14_Sequence_Listing_ST25.txt, created Mar. 13, 2020, and
having 6 KB of data.
BACKGROUND
1. Field
[0003] The disclosure of the present patent application relates to
anti-RNA virus peptides, and particularly to a MERS-CoV inhibitor
peptides.
2. Description of the Related Art
[0004] Middle East Respiratory Syndrome Coronavirus (MERS-CoV),
alternatively called HCoV-EMC/2012, causes severe respiratory
illness with symptoms including fever, cough, and shortness of
breath. Some subjects also experience diarrhea, nausea, or
vomiting. MERS-CoV is fatal for 3-4 of every ten people infected.
Currently, there is no vaccine approved to prevent transmission of
MERS-CoV and there is no specific antiviral treatment suggested for
MERS-CoV infection. Thus, preventative measures generally involve
routine avoidance of behaviors likely to lead to infection (hand
washing, covering the nose and mouth when sneezing, avoiding
contact with the eyes, nose, or mouth, and avoiding direct contact
with infected individuals). Care may include general medical
support for basic vital organ function, but does not include any
medications targeting MERS-CoV specifically.
[0005] MERS-CoV is an enveloped virus, which means a viral envelope
protein must identify a host receptor and initial membrane fusion
in order for the virus to enter and infect host cells (membrane
fusion may be either at the plasma membrane or in endosomes after
endocytosis). MERS-CoV accomplishes membrane fusion through
interactions between the virus'S-protein and host marker CD26.
[0006] Recent work in this field has focused on developing
monoclonal antibodies to MERS-CoV, or screening pre-existing small
molecule libraries to look for compounds that inhibit S-protein
mediated membrane fusion. In addition, a peptide sequence found in
the HR2 region of wild type MERS-CoV has been shown to have some
limited inhibitory effect on MERS-CoV membrane fusion. However,
this prior work has yet to deliver a pharmaceutical capable of
either preventing MERS-CoV infection or treating an infected
subject.
[0007] Thus, MERS-CoV inhibitor peptides solving the aforementioned
problems are desired.
SUMMARY
[0008] The MERS-CoV inhibitor peptides include a set of peptides
designed by modification or mutation of a wild type MERS-CoV fusion
protein. The MERS-CoV inhibitor peptides are capable of inhibition
of MERS-CoV infection in cells and may be used to prevent and/or
treat MERS-CoV infection. The MERS-CoV inhibitor peptides may also
be used as reagents for MERS-CoV inhibition assays as a standard or
reference inhibitors.
[0009] An embodiment of the present subject matter is directed to a
pharmaceutical composition including one or more of the MERS-CoV
inhibitor peptides and a pharmaceutically acceptable carrier.
[0010] An embodiment of the present subject matter is directed to a
method of making a pharmaceutical composition including mixing one
or more of the MERS-CoV inhibitor peptides under sterile conditions
with a pharmaceutically acceptable carrier and preservatives,
buffers, or propellants to create the pharmaceutical composition;
and providing the pharmaceutical composition in a form suitable for
daily, weekly, or monthly administration.
[0011] An embodiment of the present subject matter is directed to
compositions including one or more of the MERS-CoV inhibitor
peptides and one or more expression systems. The expression system
may be a viral based expression system, a plasmid based expression
system, or any other expression system suitable for causing or
enhancing expression of the MERS-CoV inhibitor peptides in a
bacterium, yeast, or mammalian cell. The expression system may
include a promoter sequence and DNA or RNA encoding one or more of
the MERS-CoV inhibitor peptides.
[0012] An embodiment of the present subject matter is directed to
methods of inhibiting MERS-CoV infection, preventing MERS-CoV
transmission, and/or treating a MERS-CoV infection, including
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition according to the
present subject matter. In a further embodiment, the methods of
inhibiting MERS-CoV infection may include preventing MERS-CoV
infection of a cell.
[0013] An embodiment of the present subject matter is directed to
methods of using the MERS-CoV inhibitor peptides as reference
agents to evaluate inhibition by other candidates against MERS CoV.
These methods may include using the MERS-CoV inhibitor peptides as
reference agents in Cell-Cell Fusion Assays, Viral Plaque Formation
Assays, Viral RNA Quantitation Assays, or the like.
[0014] These and other features of the present subject matter will
become readily apparent upon further review of the following
specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 depicts a sequence alignment of Peptides 1-12 (SEQ ID
NOs:1-12 respectively) and of the consensus sequence (SEQ ID NO:
13) and displays the % conservation of each amino acid in the
aligned sequences.
[0016] FIG. 2A depicts a graph of the results of a Cell-Cell Fusion
Assay measuring the inhibitory properties of Peptides 1-12 at 0.1,
1, and 10 .mu.M.
[0017] FIG. 2B depicts a graph of the results of a Co-Transfection
assay indicating that the Peptides have no interaction with the
Cell-Cell Fusion Assay system.
[0018] FIGS. 3A-30 depict the results of a Plaque Reduction Assay
for MERS-CoV treated with 10 .mu.M Peptides 1-12.
[0019] FIG. 4A depicts the results of a Plaque Reduction Assay for
MERS-CoV treated with Peptide 4 at 50 .mu.M, 25 .mu.M, 12.5 .mu.M,
6.25 .mu.M, and 3.125 .mu.M.
[0020] FIG. 4B depicts the results of a Plaque Reduction Assay for
MERS-CoV treated with Peptide 5 at 50 .mu.M, 25 .mu.M, 12.5 .mu.M,
6.25 .mu.M, and 3.125 .mu.M.
[0021] FIG. 4C depicts the results of a Plaque Reduction Assay for
MERS-CoV treated with Peptide 6 at 50 .mu.M, 25 .mu.M, 12.5 .mu.M,
6.25 .mu.M, and 3.125 .mu.M.
[0022] FIG. 5A depicts a graph of the effect of various
concentrations of Peptide 4 on the growth of Vero cells.
[0023] FIG. 5B depicts a graph of the effect of various
concentrations of Peptide 5 on the growth of Vero cells.
[0024] FIG. 5C depicts a graph of the effect of various
concentrations of Peptide 6 on the growth of Vero cells.
[0025] Similar reference characters denote corresponding features
consistently throughout the attached drawings.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] The MERS-CoV inhibitor peptides include a set of peptides
designed by modification or mutation of a wild type MERS-CoV fusion
protein. The MERS-CoV inhibitor peptides are capable of inhibition
of MERS-CoV infection in cells and may be used to prevent and/or
treat MERS-CoV infection. The MERS-CoV inhibitor peptides may also
be used as reagents for MERS-CoV inhibition assays as a standard or
reference inhibitors.
[0027] Throughout this application, the term "about" may be used to
indicate that a value includes the standard deviation of error for
the composition, device or method being employed to determine the
value.
[0028] The use of the term "or" in the specification and claim(s)
is used to mean "and/or" unless explicitly indicated to refer to
alternatives only or the alternatives are mutually exclusive,
although the disclosure supports a definition that refers to only
alternatives and "and/or."
[0029] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, un-recited elements or method steps. In certain
cases, the term "comprising" may be replaced with "consisting
essentially of" or "consisting of."
[0030] The use of the word "a" or "an" when used herein in
conjunction with the term "comprising" in the claims and/or the
specification may mean "one," but it is also consistent with the
meaning of "one or more," "at least one," and "one or more than
one."
[0031] The phrase "pharmaceutically acceptable," as used herein,
refers to molecular entities and compositions that do not produce
an allergic or similar untoward reaction when administered to a
human.
[0032] The term "subject," as used herein, means a mammal,
including but not limited to a human being.
[0033] As used herein, the term "providing" an agent is used to
include "administering" the agent to a subject.
[0034] As used herein, a "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, isotonic and
absorption delaying agents, buffers, carrier solutions,
suspensions, colloids, excipients, and the like.
[0035] An embodiment of the present subject matter is directed to a
pharmaceutical composition comprising one or more of the MERS-CoV
inhibitor peptides and a pharmaceutically acceptable carrier.
[0036] An embodiment of the present subject matter is directed to a
method of making a pharmaceutical composition including mixing one
or more of the MERS-CoV inhibitor peptides with a pharmaceutically
acceptable carrier. For example, the method of making a
pharmaceutical composition can include mixing a MERS-CoV inhibitor
peptide under sterile conditions with a pharmaceutically acceptable
carrier with preservatives, buffers, and/or propellants to create
the pharmaceutical composition.
[0037] An embodiment of the present subject matter is directed to a
pharmaceutical composition including one or more of the MERS-CoV
inhibitor peptides. To prepare the pharmaceutical composition, one
or more of the MERS-CoV inhibitor peptides, as the active
ingredient, are intimately admixed with a pharmaceutically
acceptable carrier according to conventional pharmaceutical
compounding techniques. Carriers are inert pharmaceutical
excipients, including, but not limited to, binders, suspending
agents, lubricants, flavorings, sweeteners, preservatives, dyes,
and coatings. In preparing compositions in oral dosage form, any of
the pharmaceutical carriers known in the art may be employed. For
example, for liquid oral preparations, suitable carriers and
additives include water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents, and the like. Further, for solid
oral preparations, suitable carriers and additives include
starches, sugars, diluents, granulating agents, lubricants,
binders, disintegrating agents, and the like.
[0038] The present compositions can be in unit dosage forms such as
tablets, pills, capsules, powders, granules, ointments, sterile
parenteral solutions or suspensions, metered aerosol or liquid
sprays, drops, ampules, auto-injector devices or suppositories, for
oral parenteral, intranasal, sublingual or rectal administration,
or for administration by inhalation or insufflation. One or more of
the MERS-CoV inhibitor peptides can be mixed under sterile
conditions with a pharmaceutically acceptable carrier and, if
required, any needed preservatives, buffers, or propellants. The
composition can be presented in a form suitable for daily, weekly,
or monthly administration. The pharmaceutical compositions herein
will contain, per dosage unit, e.g., tablet, capsule, powder,
injection, teaspoonful, suppository and the like, an amount of the
active ingredient necessary to deliver an effective dose. A
therapeutically effective amount of a MERS-CoV inhibitor peptide or
an amount effective to treat a disease, such as a coronavirus
infection, may be determined initially from the Examples described
herein and adjusted for specific targeted diseases using routine
methods.
[0039] An embodiment of the present subject matter is directed to
compositions including one or more of the MERS-CoV inhibitor
peptides and one or more expression systems. The expression system
may be a viral based expression system, a plasmid based expression
system, or any other expression system suitable for causing or
enhancing expression of the MERS-CoV inhibitor peptides in a
bacterium, yeast, or mammalian cell. The expression system may
include a promoter sequence and DNA or RNA encoding one or more of
the MERS-CoV inhibitor peptides.
[0040] An embodiment of the present subject matter is directed to
methods of using the MERS-CoV inhibitor peptides as reference
agents to evaluate inhibition by other candidates against MERS CoV.
These methods may include using the MERS-CoV inhibitor peptides as
reference agents in Cell-Cell Fusion Assays, Viral Plaque Formation
Assays, Viral RNA Quantitation Assays, or the like.
[0041] The MERS-CoV inhibitor peptides can be administered to a
subject in need thereof. In an embodiment, the MERS-CoV inhibitor
peptides can be administered to a subject in need thereof to
inhibit MERS-CoV infection, preventing MERS-CoV transmission,
and/or treating a MERS-CoV infection.
[0042] An embodiment of the present subject matter is directed to a
method of inhibiting MERS-CoV infection, preventing MERS-CoV
transmission, and/or treating a MERS-CoV infection, comprising
administering to a subject in need thereof a therapeutically
effective amount of the pharmaceutical composition according to the
present subject matter.
[0043] The MERS-CoV inhibitor peptides or pharmaceutical
compositions thereof can be administered to a subject by any
suitable route. For example, the compositions can be administered
nasally, rectally, intracisternally, intraperitoneally,
transdermally (as by powders, ointments, or drops), and/or
parenterally. As used herein, "parenteral" administration refers to
modes of administration other than through the gastrointestinal
tract, which include intravenous, intramuscular, intraperitoneal,
intrasternal, intramammary, intraocular, intrapulmonary,
intrathecal, subcutaneous and intraarticular injection and
infusion. Surgical implantation may also be contemplated,
including, for example, embedding a composition of the disclosure
in the body such as, for example, in a tissue, in the abdominal
cavity, under the splenic capsule, brain, or in the cornea.
[0044] Accordingly, the route of administration can include
intranasal administration, oral administration, inhalation
administration, subcutaneous administration, transdermal
administration, intradermal administration, intra-arterial
administration with or without occlusion, intracranial
administration, intraventricular administration, intravenous
administration, buccal administration, intraperitoneal
administration, intraocular administration, intramuscular
administration, implantation administration, topical
administration, intratumor administration, and/or central venous
administration.
[0045] The MERS-CoV inhibitor peptides are designed by modification
or mutation of a surface structure protein of MERS-CoV in the virus
S2 spike region. The heptad repeat regions (HR1 and HR2) of S2
interact to help in fusion of MERS-CoV with cell membranes. The
MERS-CoV inhibitor peptide S2 HR2 derivatives were optimized to
interfere with the proper mechanism of HR1-HR2 interactions.
[0046] The following examples illustrate the present subject
matter.
Example 1
Synthesis of MERS-CoV Inhibitor Peptides
[0047] The sequence of the HR2 region of wild type MERS-CoV is
reported in Table 1 as Peptide 1 (SEQ ID NO: 1). This peptide was
synthesized and used in assays as a reference or standard against
which the activity of new peptides was compared.
[0048] To optimize a new sequence related to MERS-CoV HR2 with
improved binding potency, computational studies were implemented to
generate a set 684 potential candidates by a series of mutations.
Free energy-based optimization was computationally carried out to
yield a set of top suggested point mutations. Several point
mutations were then generated in different combinations with
estimated improved binding strength and stability after testing by
molecular dynamics experiments. Finally, a set of 11 peptides were
synthesized (peptides 2-12 in Table 1).
[0049] Several systemic point mutations for every residue in the
wild type peptide were generated. Mutations were performed by
replacing each amino acid with any other member of the 21 known
essential amino acids. All mutations were generated only in
MERS-CoV HR2. After each point mutation, the free energy of binding
of HR1 and mutated HR2 was calculated as previously described.
(Dehouck, Y. et al., "BeAtMuSiC: Prediction of changes in
protein-protein binding affinity on mutations," Nucleic Acids
Research, 41: pp. W333-W339 (2013)). Candidates with the highest
values of binding free energy were synthesized for Anti-MERS-CoV
testing (FIG. 1 and Table 1). To confirm binding stability of each
peptide after mutation, every HR1-HR2 complex was subjected to a
molecular dynamics simulation as previously described. (Kandeel, M.
et al., "Molecular dynamics of Middle East Respiratory Syndrome
Coronavirus (MERS-CoV) fusion heptad repeat trimers," Computational
Biology and Chemistry 75: pp. 205-212 (2018)).
[0050] A set of 12 peptides were obtained by custom synthesis
service order to Biomatik Inc (Cambridge, ON, Canada) (Table 1).
The peptides were HPLC purified and confirmed by mass spectrum to
ensure maximum purity and removal of chemicals and byproducts
during the peptide synthesis process.
TABLE-US-00001 TABLE 1 The sequence of wild type and designed
peptides (mutations in bold) Name Peptide Sequence SEQ ID NUMBER
Peptide 1 (WT) SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL SEQ ID NO: 1
Peptide 2 SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKHL SEQ ID NO: 2 Peptide
3 SLTQINTTLLDLTYEMKSLQQVVKALNESYIDLKEL SEQ ID NO: 3 Peptide 4
SLTQINWTLLDLTYEMESLQQVVKALNESYIDLKEL SEQ ID NO: 4 Peptide 5
SLTQINWTLLDLTYEMESLQQVVKALNEYYIDLKEL SEQ ID NO: 5 Peptide 6
SLTQINWILLDLTYEMESLQQVVKALNEYYIDLKHL SEQ ID NO: 6 Peptide 7
SLTQINWTLLDLTYEMESLQQVMKALNEYYIDLKHL SEQ ID NO: 7 Peptide 8
SLTQINTTLLDLEYEMLSLQQVVKALNESYIDLKEL SEQ ID NO: 8 Peptide 9
SLTQINTTLLDLEYEMRSLQQVVKALNESYIDLKEL SEQ ID NO: 9 Peptide 10
SLTQINTTLLDLEYEMRSLEEVVKALNESYIDLKEL SEQ ID NO: 10 Peptide 11
SLTQINTTLLDLEYEMRSLEEVVKKLNESYIDLKEL SEQ ID NO: 11 Peptide 12
SLTQINTTLLDLEYEMRSLEEVVKKLNESYIDEKEL SEQ ID NO: 12
Example 2
Cell-Cell Fusion Assay of MERS-CoV Inhibitor Peptides
[0051] Cell-cell fusion assays were performed to quantitate the
cell-cell fusion as described previously. (Yamamoto, M. et al.,
"Identification of Nafamostat as a Potent Inhibitor of Middle East
Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion
Using the Split-Protein-Based Cell-Cell Fusion Assay," Antimicrob.
Agents Chemother. 60(11): pp. 6532-6539 (2016)). Briefly, a pair of
293FT-based reporter cells, effector and target cells, that express
individual split reporters (DSP1-7 and DSP8-11 proteins) were used,
because DSP1-7 and DSP8-11 produce fluorescence and luminescence
only when the two proteins form a tight complex. Effector cells
stably expressing DSP8-11 and S-protein and target cells stably
expressing DSP1-7 together with CD26 and TMPRSS2 were prepared. Two
hours before the fusion assay, both effector and target cells were
treated with 6 .mu.M EnduRen (Promega, Madison, Wis., USA), a
substrate for Renilla luciferase, to activate EnduRen. Each peptide
was dissolved in 10% dimethyl sulfoxide (DMSO) and added to
384-well plates (Greiner Bioscience, Frickenhausen, Germany) using
a 12-stage workstation (Biotech, Tokyo, Japan). Next, a Multidrop
dispenser (Thermo Scientific, Waltham, Mass., USA) was used to add
50 .mu.l of each single cell suspension (1.5.times.10.sup.4
effector and target cells) to the wells. Incubation was performed
at 37.degree. C. for h, then RL activity measurements were obtained
with a microplate reader (PHERAStar Plus, BMG Labtech, Cary, N.C.,
USA).
[0052] The results indicated strong inhibition of cell-cell fusion
at 10 and 1 .mu.M (FIG. 2A). The most potent peptides were peptides
5, 8, 10 and 11, with EC50 values in low and middle nanomolar range
(Table 2). Peptides 5, 8, 10 and 11 showed stronger inhibition
compared with peptide 1 (the original wild type MERS-CoV sequence).
A co-transfection control assay demonstrated that the peptides did
not interact with the assay system (See FIG. 2B).
TABLE-US-00002 TABLE 2 EC50 values of peptides by using cell-cell
fusion assay Peptide EC50 (.mu.M) 1 (SEQ. ID 1.055 NO: 1) 2 (SEQ.
ID 0.916 NO: 2) 3 (SEQ. ID 0.938 NO: 3) 4 (SEQ. ID 1.492 NO: 4) 5
(SEQ. ID 0.432 NO: 5) 6 (SEQ. ID 0.917 NO: 6) 7 (SEQ. ID 2.355 NO:
7) 8(SEQ. ID 0.465 NO: 8) 9 (SEQ. ID 1.299 NO: 9) 10 (SEQ. ID 0.426
NO: 10) 11 (SEQ. ID 0.039 NO: 11) 12(SEQ. ID 0.831 NO: 12)
Example 3
Plaque Assay of MERS-CoV Inhibitor Peptides
[0053] African green monkey kidney cells (Vero cells) were
purchased from the American Type Culture Collection (ATCC,
Manassas, Va., USA). The cell culture was kept in a CO2 incubator
at 37.degree. C. in Dulbecco's modified Eagle's medium (DMEM,
Thermo Fisher Scientific, Waltham, Mass., USA) containing 10% fetal
bovine serum (FBS, Thermo Fisher Scientific), 25 mM HEPES, 100 U/ml
penicillin and 100 .mu.g/ml streptomycin. MERS-CoV was obtained
from the Korea Centers for Disease Control and Prevention
(CoV/KOR/KNIH/002_05_2015, Permission No.
1-001-MER-IS-2015001).
[0054] The plaque reduction assay was performed as reported
previously. (Park, B. K. et al., "Generation and characterization
of a monoclonal antibody against MERS-CoV targeting the
spike-protein using a synthetic peptide epitope-CpG-DNA-liposome
complex," BMB Rep. 52(6): pp. 397-402 (2019)). Briefly, Vero cells
were cultivated on six-well plates for 12 h at 6.times.10.sup.5
cells/well. In an initial study, MERS-CoV was mixed with each
peptide at a final concentration of 10 .mu.M for 30 min at
37.degree. C. The mixtures of MERS-CoV and each peptide were added
to Vero cells in each well and then incubated for 1 h. After
incubation, the supernatants were removed and DMEM/F12 medium
(Thermo Fisher Scientific) containing 0.6% oxoid agar was
transferred to each well. Four days after infection, plaque
formation was observed by staining with crystal violet and plaque
numbers were counted (See FIGS. 3A-30). The initial test results
revealed strong inhibition of MERS-CoV by peptides 4, 5, and 6. The
other peptides produced a more moderate decrease in plaque
formation. Notable results include 98.3% inhibition of plaque
formation by Peptide 6, 98.2% inhibition by Peptide 4, 95%
inhibition by Peptide 5, 74% inhibition by Peptide 2, and 69-70%
inhibition by Peptides 11 and 12. (See Table 3)
TABLE-US-00003 TABLE 3 Plaque Inhibition Assay of Peptides 1-12
Percent of Sample # Plaques Normalize DMSO # Control 142 568
92.20779 DMSO 154 616 100 Peptide 1 55 220 35.71429 (SEQ ID NO: 1)
Peptide 2 36 144 23.37662 (SEQ ID NO: 2) Peptide 3 55 220 35.71429
(SEQ ID NO: 3) Peptide 4 11 11 1.785714 (SEQ ID NO: 4) Peptide 5 31
31 5.032468 (SEQ ID NO: 5) Peptide 6 8 8 1.298701 (SEQ ID NO: 6)
Peptide 7 55 220 35.71429 (SEQ ID NO: 7) Peptide 8 92 368 59.74026
(SEQ ID NO: 8) Peptide 9 81 324 52.5974 (SEQ ID NO: 9) Peptide 10
59 236 38.31169 (SEQ ID NO: 10) Peptide 11 46 184 29.87013 (SEQ ID
NO: 11) Peptide 12 48 192 31.16883 (SEQ ID NO: 12)
[0055] After initial confirmation of the inhibitory properties of
the peptides, Peptides 4, 5, and 6 were tested in varying
concentrations, ranging from 50 .mu.M to 3.125 .mu.M (See FIGS.
4A-4C). The results of these experiments were then used to
calculate the half maximal effective concentration for Peptides 4,
5, and 6. The results are provided in Table 4.
TABLE-US-00004 TABLE 4 EC50 of Peptides 4, 5, and 6. Sample EC50
(.mu.M) Peptide 4 0.302 Peptide 5 1.428 Peptide 6 1.849
Example 4
Cytotoxicity and Viability
[0056] Vero cells (1.times.10.sup.3 per well) were plated on
96-well plates and cultured for 12 h. The cells were treated with
three fold serial dilutions of Peptides 4, 5, or 6, or with 10%
DMSO (control) for 3 days (Peptide concentrations ranged from 100
.mu.M to 0.4 .mu.M). Then, cells were treated with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Sigma-Aldrich, St. Louis, Mo., USA) for 4 h at 37.degree. C.
Formazan crystals were dissolved in DMSO, and the absorbance at 570
nm was measured using a microplate reader (Thermo Fisher
Scientific, Ratastie, Finland).
[0057] No cytotoxicity at concentrations equal to or lower than 10
.mu.M was observed for any of the tested peptides (See FIGS.
5A-5C). Thus, it was concluded that Peptides 4, 5 and 6 have a safe
cellular profile.
[0058] It is to be understood that the MERS-CoV Inhibitor Peptides
are not limited to the specific embodiments described above, but
encompasses any and all embodiments within the scope of the generic
language of the following claims enabled by the embodiments
described herein, or otherwise shown in the drawings or described
above in terms sufficient to enable one of ordinary skill in the
art to make and use the claimed subject matter.
Sequence CWU 1
1
13136PRTMiddle East Respiratory Syndrome Coronavirus 1Ser Leu Thr
Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr Tyr Glu Met1 5 10 15Leu Ser
Leu Gln Gln Val Val Lys Ala Leu Asn Glu Ser Tyr Ile Asp 20 25 30Leu
Lys Glu Leu 35236PRTMiddle East Respiratory Syndrome Coronavirus
2Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr Tyr Glu Met1 5
10 15Leu Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu Ser Tyr Ile
Asp 20 25 30Leu Lys His Leu 35336PRTMiddle East Respiratory
Syndrome Coronavirus 3Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp
Leu Thr Tyr Glu Met1 5 10 15Lys Ser Leu Gln Gln Val Val Lys Ala Leu
Asn Glu Ser Tyr Ile Asp 20 25 30Leu Lys Glu Leu 35436PRTMiddle East
Respiratory Syndrome Coronavirus 4Ser Leu Thr Gln Ile Asn Trp Thr
Leu Leu Asp Leu Thr Tyr Glu Met1 5 10 15Glu Ser Leu Gln Gln Val Val
Lys Ala Leu Asn Glu Ser Tyr Ile Asp 20 25 30Leu Lys Glu Leu
35535PRTMiddle East Respiratory Syndrome Coronavirus 5Ser Leu Thr
Gln Ile Asn Trp Thr Leu Leu Asp Leu Thr Tyr Glu Met1 5 10 15Glu Ser
Leu Gln Gln Val Val Lys Ala Leu Asn Glu Tyr Tyr Ile Asp 20 25 30Leu
Lys Glu 35636PRTMiddle East Respiratory Syndrome Coronavirus 6Ser
Leu Thr Gln Ile Asn Trp Thr Leu Leu Asp Leu Thr Tyr Glu Met1 5 10
15Glu Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu Tyr Tyr Ile Asp
20 25 30Leu Lys His Leu 35736PRTMiddle East Respiratory Syndrome
Coronavirus 7Ser Leu Thr Gln Ile Asn Trp Thr Leu Leu Asp Leu Thr
Tyr Glu Met1 5 10 15Glu Ser Leu Gln Gln Val Met Lys Ala Leu Asn Glu
Tyr Tyr Ile Asp 20 25 30Leu Lys His Leu 35836PRTMiddle East
Respiratory Syndrome Coronavirus 8Ser Leu Thr Gln Ile Asn Thr Thr
Leu Leu Asp Leu Glu Tyr Glu Met1 5 10 15Leu Ser Leu Gln Gln Val Val
Lys Ala Leu Asn Glu Ser Tyr Ile Asp 20 25 30Leu Lys Glu Leu
35936PRTMiddle East Respiratory Syndrome Coronavirus 9Ser Leu Thr
Gln Ile Asn Thr Thr Leu Leu Asp Leu Glu Tyr Glu Met1 5 10 15Arg Ser
Leu Gln Gln Val Val Lys Ala Leu Asn Glu Ser Tyr Ile Asp 20 25 30Leu
Lys Glu Leu 351036PRTMiddle East Respiratory Syndrome Coronavirus
10Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Glu Tyr Glu Met1
5 10 15Arg Ser Leu Glu Glu Val Val Lys Ala Leu Asn Glu Ser Tyr Ile
Asp 20 25 30Leu Lys Glu Leu 351136PRTMiddle East Respiratory
Syndrome Coronavirus 11Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp
Leu Glu Tyr Glu Met1 5 10 15Arg Ser Leu Glu Glu Val Val Lys Lys Leu
Asn Glu Ser Tyr Ile Asp 20 25 30Leu Lys Glu Leu 351236PRTMiddle
East Respiratory Syndrome Coronavirus 12Ser Leu Thr Gln Ile Asn Thr
Thr Leu Leu Asp Leu Glu Tyr Glu Met1 5 10 15Arg Ser Leu Glu Glu Val
Val Lys Lys Leu Asn Glu Ser Tyr Ile Asp 20 25 30Glu Lys Glu Leu
351336PRTMiddle East Respiratory Syndrome
Coronavirusmisc_feature(17)..(17)Xaa can be any naturally occurring
amino acid 13Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr
Tyr Glu Met1 5 10 15Xaa Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu
Ser Tyr Ile Asp 20 25 30Leu Lys Glu Leu 35
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