Identification of inhibitor peptides to bind with n-terminal spike (s) and nonstructural protein (NSP) sequences of SARS-COV2 b.1.617.2 Delta or Omicron variants alone and combination with drug(s) for targeted antiviral therapy

The last 5 years literature search shows limited approach towards development of successful drug and/or inhibitor protein/peptide sequences for the virus. The research publications on SARS-COV2 inhibitors are cited have contemporary importance in context of our patent application. The bibliographic references are presented below:

ZHOU, H., YANG, J., ZHOU, C., CHEN, B., FANG, H., CHEN, S., ZHANG, X., WANG, L. & ZHANG, L. 2021. A Review of SARS-CoV2: Compared With SARS-CoV and MERS-CoV. Front Med ( Lausanne ), 8, 628370.

XIU, S., DICK, A., JU, H., MIRZAIE, S., ABDI, F., COCKLIN, S., ZHAN, P. & LIU, X. 2020. Inhibitors of SARS-CoV-2 Entry: Current and Future Opportunities. J Med Chem, 63, 12256-12274.

SCHUTZ, D., RUIZ-BLANCO, Y. B., MUNCH, J., KIRCHHOFF, F., SANCHEZ-GARCIA, E. & MULLER, J. A. 2020. Peptide and peptide-based inhibitors of SARS-CoV-2 entry. Adv Drug Deliv Rev, 167, 47-65.

CHENG, Y. W., CHAO, T. L., LI, C. L., CHIU, M. F., KAO, H. C., WANG, S. H., PANG, Y. H., LIN, C. H., TSAI, Y. M., LEE, W. H., TAO, M. H., HO, T. C., WU, P. Y., JANG, L. T., CHEN, P. J., CHANG, S. Y. & YEH, S. H. 2020. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep, 33, 108254.

KAI, H. & KAI, M. 2020. Interactions of coronaviruses with ACE2, angiotensin II, and RAS inhibitors-lessons from available evidence and insights into COVID-19 . Hypertens Res, 43, 648-654.

HIRSCH, J. S., NG, J. H., ROSS, D. W., SHARMA, P., SHAH, H. H., BARNETT, R. L., HAZZAN, A. D., FISHBANE, S., JHAVERI, K. D., NORTHWELL, C.-R. C. & NORTHWELL NEPHROLOGY, C.-R. C. 2020. Acute kidney injury in patients hospitalized with COVID-19 . Kidney Int, 98, 209-218.

DE LUCA, G., NARDIN, M., ALGOWHARY, M., UGUZ, B., OLIVEIRA, D. C., GANYUKOV, V., ZIMBAKOV, Z., CERCEK, M., JENSEN, L. O., LOH, P. H., CALMAC, L., ROURA FERRER, G., QUADROS, A., MILEWSKI, M., SCOTTO DI UCCIO, F., VON BIRGELEN, C., VERSACI, F., TEN BERG, J., CASELLA, G., LUNG, A. W. S., KALA, P., DIEZ GIL, J. L., CARRILLO, X., DIRKSEN, M., BECERRA-MUNOZ, V. M., LEE, M. K., JUZAR, D. A., DE MOURA JOAQUIM, R., PALADINO, R., MILICIC, D., DAVLOUROS, P., BAKRACESKI, N., ZILIO, F., DONAZZAN, L., KRAAIJEVELD, A., GALASSO, G., LUX, A., MARINUCCI, L., GUIDUCCI, V., MENICHELLI, M., SCOCCIA, A., YAMAC, A. H., MERT, K. U., FLORES RIOS, X., KOVARNIK, T., KIDAWA, M., MOREU, J., FLAVIEN, V., FABRIS, E., MARTINEZ-LUENGAS, I. L., BOCCALATTE, M., BOSA OJEDA, F., ARELLANO-SERRANO, C., CAIAZZO, G., CIRRINCIONE, G., KAO, H. L., SANCHIS FORES, J., VIGNALI, L., PEREIRA, H., MANZO, S., ORDONEZ, S., ARAT OZKAN, A., SCHELLER, B., LEHTOLA, H., TELES, R., MANTIS, C., ANTTI, Y., BRUM SILVEIRA, J. A., ZONI, R., BESSONOV, I., SAVONITTO, S., KOCHIADAKIS, G., ALEXOPULOS, D., URIBE, C. E., KANAKAKIS, J., FAURIE, B., GABRIELLI, G., GUTIERREZ BARRIOS, A., BACHINI, J. P., ROCHA, A., TAM, F. C., RODRIGUEZ, A., LUKITO, A. A., SAINT-JOY, V., PESSAH, G., TUCCILLO, A., CORTESE, G., PARODI, G., BOURAGHDA, M. A., KEDHI, E., LAMELAS, P., SURYAPRANATA, H. & VERDOIA, M. 2021. Renin-angiotensin system inhibitors and mortality among diabetic patients with STEMI undergoing mechanical reperfusion during the COVID-19 pandemic. Diabet Epidemiol Manag, 4, 100022.

KURBEL, S. 2021. The renin-angiotensin system in COVID-19: Why ACE2 targeting by coronaviruses produces higher mortality in elderly hypertensive patients? Bioessays, 43, e2000112.

DASGUPTA, S. & BANDYOPADHYAY, M. 2021. Molecular docking of SARS-COV-2 Spike epitope sequences identifies heterodimeric peptide-protein complex formation with human Zo-1, TLR8 and brain specific glial proteins. Med Hypotheses, 157, 110706.

ARTESE, A., SVICHER, V., COSTA, G., SALPINI, R., DI MAIO, V. C., ALKHATIB, M., AMBROSIO, F. A., SANTORO, M. M., ASSARAF, Y. G., ALCARO, S. & CECCHERINI-SILBERSTEIN, F. 2020. Current status of antivirals and druggable targets of SARS CoV-2 and other human pathogenic coronaviruses. Drug Resist Updat, 53, 100721.

BANERJEE, R., PERERA, L. & TILLEKERATNE, L. M. V. 2021. Potential SARS-CoV-2 main protease inhibitors. Drug Discov Today, 26, 804-816.

CITARELLA, A., SCALA, A., PIPERNO, A. & MICALE, N. 2021. SARS-CoV-2 M(pro): A Potential Target for Peptidomimetics and Small-Molecule Inhibitors. Biomolecules, 11.

FISCHER, A., SELLNER, M., MITUSINSKA, K., BZOWKA, M., LILL, M. A., GORA, A. & SMIESKO, M. 2021. Computational Selectivity Assessment of Protease Inhibitors against SARS-CoV-2 . Int J Mol Sci, 22.

SABBAH, D. A., HAJJO, R., BARDAWEEL, S. K. & ZHONG, H. A. 2021. An Updated Review on SARS-CoV-2 Main Proteinase (M(Pro)): Protein Structure and Small-Molecule Inhibitors. Curr Top Med Chem, 21, 442-460.

SACCO, M. D., MA, C., LAGARIAS, P., GAO, A., TOWNSEND, J. A., MENG, X., DUBE, P., ZHANG, X., HU, Y., KITAMURA, N., HURST, B., TARBET, B., MARTY, M. T., KOLOCOURIS, A., XIANG, Y., CHEN, Y. & WANG, J. 2020. Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against M(pro) and cathepsin L. Sci Adv, 6.

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Not applicable.

The invention research did not receive any fund from any federally sponsored or private Grantor organizations.

Not applicable.

The Severe Acute Respiratory Syndrome (SARS) Corona virus 2 (SARS-COV2) is the etiological agent of recent pandemic (COVID-19) (end of 2019 till 2021 and continuing). The search for the origin of COVID-19 indicated that the virus has zoonotic origin and found to infect the people of Wuhan, China first. The COVID-19 infection has close similarity with Influenza virus as it starts with blank cough, headache, upper respiratory Tract discomfort to lower respiratory tract illness pneumonia, respiratory distress, asphyxia with fever ended up to coma. The point-source infection eventually turned into global pandemic mostly through international traveling all over the world.

Though, there is a relationship between the recent SARS-COV2 with other closely related Corona viruses like, MERS-CoV, HCoV-229E, HCoV-HKU1, HCoV-NL63, HCoV-0C43 (1) which prevail since a decade at least, there is no successful therapeutic interventions developed to prevent virus spread.

SARS-CoV, MERS-CoV and SARS-COV2 are under genus betacoronavirus which infect birds and bat as primary hosts. The mechanism of infectivity and severity of SARS-COV2 in human depends on (a) entry of virus; (b) replication mechanism of virus within host cells; (c) escape mechanism of progeny viruses from hosts' immune response mediated killing process; (d) genetic susceptibility of hosts and (e) mutation in virus Spike and replicase proteins. Recent advancement shows spread of mutant virus variants (variant of concerns): Delta, Kappa, Omicron in certain parts of the world including United States. These variants have considerable numbers of mutations in their single stranded RNA genome which develop new viral protein sequences. As found Delta variant was more virulent than Omicron. The reservoir, if any, for the viruses, other than infected human beings, is still unknown. Still, it can be predicted closely that, infected domestic animals, birds can act as reservoirs for these variants in the communities where we find spread of infection.

The treatment is still obscure for COVID-19, only experimental medications and supportive treatments are there rather than targeted antiviral therapy. The recent outcome of RNA vaccines has limited success to reduce SARS-COV2 infection. The vaccines are also expected to reduce Delta and Omicron infection.

Recent investigations demonstrate inhibitors to block SARS-COV2 entry (2-4). This is one of the current aspects of immunotherapy approaches besides vaccine preparation. Available reports published in NCBI PubMed, and Google Scholar sites indicate viruses are found to attach with Angiotensin Converting Enzyme 2 (ACE2) and therefore modify blood pressure level and causes hypertension with acute kidney infection (5, 6). However, investigations demonstrate variable outcomes of ACE2 inhibition in different age groups and preexisting disease conditions like diabetes (7, 8). Our molecular dynamics and docking experiments demonstrate different non-ACE2 proteins like innate immune responder Toll-like receptor-8 (TLR8), tight junction protein Zonula Occludens (Zo), glial activation protein NDRG2, Apo S100B have strong propensity to bind with SARS-COV2 Spike protein derived peptides (9).

The current research indicates role of protease inhibitors in attenuation of SARS-COV2 infection. Several investigations demonstrated role of viral main protease (Mpro) during infection and propose to develop drug and peptide blockers (10-18).

In the patent application, we provide the invented inhibitor peptide sequences developed from immunodominant proteins of SARS-COV2 (B.1.617.2) and its Delta, Omicron variants.

In continuation of our research, we screened immunodominant B cell epitope—peptides (antibody binding sites) from SARS-COV2 Delta Variant (B.1.617.2) Spike protein N-terminal sequences (7ORB_R, 7SOA_A) by using IEDB epitope Tool (NIAID) and took these virus peptides as templates to construct peptide inhibitors aimed to bind with specific sequences of Spike glycoprotein to prevent virus entry. Also, we identified inhibitor sequences for Open Reading Frame 1ab (ORF1ab) to target virus nonstructural protein sequences (NSP1-14). The binding parameters are determined from the closest and overlapping position of inhibitor and virus peptide sequences to construct stable heterodimeric structures. The Dimer Packing Quality Values (FSCOR) are obtained from each set of dimeric structure by Preddimer NMR structure analysis software showing highest FSCOR value is the best dimeric structure conformation. We also evaluated specificity of the identified inhibitors to bind SARS-COV2 Delta variant (B.1.617.2) and related protein sequences. Our observations show all these inhibitors have different degrees of binding efficiency including the strongest to moderate to minimum abilities to form heterodimer complexes. Thus, we provide evidence of new peptide inhibitors to block functions of immunodominant regions of (a) Spike envelope protein and (b) nonstructural proteins (NSP1-12) of SARS-COV2 and its Delta variants including Omicron. The patent application includes invented inhibitor peptide sequences against immunodominant protein sequences of SARS-COV2 (B.1.617.2) including Delta and Omicron variants.

8. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF DRAWING(S)

FIG. 1 . Molecular Docking and digital analysis of binding between inhibitor peptide ISD1 (chocolate brown ball and stick structure) [SEQ1] and SARS-COV2 (B.1.617.2) Delta variant N-terminal Spike S1 protein 70RB_R sequence (Tertiary protein structure; alpha helix: red and pleated structure: yellow).

FIG. 2 . Molecular Docking and digital analysis of binding between inhibitor peptide ISD3 (green ball and stick structure) [SEQ3] and SARS-COV2 (B.1.617.2) Delta variant N-terminal Spike S1 protein 70RB_R sequence (Tertiary protein structure; alpha helix: red and pleated structure: yellow).

FIG. 3 . Molecular Docking and digital analysis of binding between inhibitor peptide SO11(blue ball and stick structure) [SEQ14] and SARS-COV2 (B.1.617.2) Delta variant N-terminal Spike S1 protein sequence (SOA1: 3D atomic structure) [SEQ28].

FIG. 4 . Molecular Docking and digital analysis of binding between inhibitor peptide S04 [SEQ51](deep green ball and stick structure) and SARS-COV2 (B.1.617.2) Delta variant N-terminal Spike S1 protein sequence (SOA4: 3D atomic structure) [SEQ31].

FIG. 5 . Molecular Docking and digital analysis of binding between ORF1 ab inhibitor peptide lp9L (pink ball and stick structure) [SEQ13] and SARS-COV2 (B.1.617.2) Delta variant Open Reading Frame ORflab selected protein sequence (protein sequence Ep9L [SEQ49]: 3D atomic structure).

FIG. 6 . Molecular Docking and digital analysis of binding between inhibitor peptide lp2L [SEQ7](green ball and stick structure), Ip4L [SEQ52] (purple ball stick model) and SARS-COV2 (B.1.617.2) ORF1ab sequence EP2L [SEQ42].

FIG. 7 . Molecular docking experiment demonstrates binding of antiviral drug Remdesivir and peptide inhibitor S05 [SEQ16] combination with SARS-COV2 B.1.617.2 Delta/Omicron Spike protein 70RB_R (Remdesivir (CID 56832906): Green 3D atomic structure; S05 [SEQ16]: ball and stick and 70RB_R: tertiary protein structure).

FIG. 8 . Molecular docking experiment demonstrates binding of anti-HIV drug Efavirenz (ID: CHEMBL1309, PubChem) and identified peptide inhibitor lp9L (green ball and stick structure) [SEQ13] combination with SARS-COV2 B.1.617.2 Delta/Omicron Open Reading Frame ORF1ab (NSP1-14) truncated peptide sequence E10L [SEQ50] (Efavirenz: purple 3D atomic structure; E10L: 3D atomic structure).

A. Protein Sequence Selection.

We selected SARS-COV2 (B.1.617.2) protein sequences from NCBI, Protein database. The sequences are, 7ORB_R 1-205 amino acid sequence); 7SOA_A (SARS coronavirus Tor2), Sequence ID: NP_828849.7 (7073 amino acid).

The selected virus protein sequences are transformed into corresponding FASTA sequences by using NCBI PubMed software system.

The FASTA sequences were fed individually to IEDB Tools linear B cell epitope identification software (Bepipred linear epitope prediction tool, IEDB).

The immunodominant sequences which exhibit potential antibody binding sites and amino acids are selected for construction of peptide inhibitor.

Random shuffling of amino acids was performed to construct each identified peptide inhibitors corresponding to immunodominant virus protein sequences.

The inhibitor peptide sequences, and corresponding virus peptides obtained from protein sequences are named to identify them individually and kept for record.

B. Experiments to Determine Efficacy of Invented Inhibitor Peptides

The physical parameters like hydrophobicity values, pH/isoelectric point of the peptide, Mass, Extinction coefficient, polarity of amino acids in peptide sequence are determined by using chemistry software pepDraw (USA) and Avogadro (USA). The 30 amino acid inhibitor peptide sequences were then chosen for determining their efficacy to form heterodimeric structures with viral peptide sequences at the best fit to least fit orders at pH7-7.2. We use Preddimer software for transmembrane membrane alpha helical protein sequence interaction study purposes.

C. Results of the Experiments

The results are presented in the Tables 1, 2 and 3. The Preddimer heterodimer identification software demonstrated several models. The higher values of FSCOR (Dimer Packing Quality) are chosen as determining value for the best fit heterodimers thus confirm the binding affinity of inhibitor peptides with viral protein sequences for neutralization purposes. The PDB format of all the invented Inhibitor sequences and immunodominant virus peptides are constructed to determine the three-dimensional interaction pattern between them. We chose molecular docking and molecular dynamics experiments to find out confirmation of inhibitor peptide interaction with viral protein sequences and visualize.

The term, Pack, indicates relative number of atoms within the structure; the term, Int, is the function accounting complementarity of hydrophobic properties on the helix-helix interface. The term, Env, is estimating correspondence of polar and structural properties of the dimer surface outside membrane lipid environment. The function (Int): (α1, α2, X, R, d); R is the distance between helical axes and d is the shift along the helical axis. The higher value of F SCOR indicates the best position of the stable dimer formed between the inhibitor and Spike peptides.

The term, Pack, indicates relative number of atoms within the structure; the term, Int, is the function accounting complementarity of hydrophobic properties on the helix-helix interface. The term, Env, is estimating correspondence of polar and structural properties of the dimer surface outside membrane lipid environment. The function (Int): (α1, α2, X, R, d); R is the distance between helical axes and d is the shift along the helical axis. The higher value of FSCOR indicates the best position of the stable dimer formed between the inhibitor and Spike peptides.

The term, Pack, indicates relative number of atoms within the structure; the term, Int, is the function accounting complementarity of hydrophobic properties on the helix-helix interface. The term, Env, is estimating correspondence of polar and structural properties of the dimer surface outside membrane lipid environment. The function (Int): (α1, α2, X, R, d); R is the distance between helical axes and d is the shift along the helical axis. The higher value of F SCOR indicates the best position of the stable dimer formed between the inhibitor and Spike peptides.