LifeTein synthesized a long peptide of 75 residues: C subunit of the ATP synthase

LifeTein synthesized the C subunit of the ATP synthase. The c subunit is a highly hydrophobic peptide of 75 residues. This is a transmembrane α-helical “hairpin” localized in the mitochondrial inner membrane where it assembles into oligomers of 8–16 units depending on the species.

The long peptide of the C subunit spontaneously folds into a β‑sheet conformation. This was confirmed by the peptide’s far-UV circular dichroism (CD) spectrum. The c subunit in β-sheet conformation could form aggregates. The formation of cross-β aggregates was monitored using the amyloid-binding dye Tiofavin T (TT). It was found that a critical role for Ca2+ in the folding and self-assembly of c subunits into oligomers rather than fibrils.

The amyloidogenic peptides can form ion channels in lipid bilayers. The human unmodified synthetic c subunit is an amyloidogenic peptide and its oligomers
are capable of forming ion-conducting pores in planar lipid bilayers. Similarly, amyloid and synuclein in their oligomeric conformations can form ion channels in planar lipid bilayers with lower conductances compared to the canonical PTP. These toxic forms of misfolded c subunit might play a significant role in cell pathophysiology.

Reference: https://www.nature.com/articles/s41598-021-88157-z.pdf

Long Peptide Synthesis of C Subunit

Rabbit Polyclonal Anti-gM and Anti-gN Antibodies Using Synthetic Long Peptides

Human cytomegalovirus (HCMV) is currently a major cause of congenital disease in newborns and organ failure in transplant recipients. Various vaccine strategies have been developed, including live attenuated, recombinant viral proteins, dense bodies, vector vaccine subunit, or synthetic peptide epitopes. The rabbit polyclonal anti-gM and anti-gN antibodies were elicited by vaccinating animals with synthesized peptide sequences of gM (1-13aa and 345-372aa) and gN (61-101aa). Animal immunization and sera collection were performed at Lifetein, LLC (Hillsborough, NJ, USA). The sera were affinity-purified by its corresponding peptides.

These formulations have been extensively tested using different animal models and have shown promising immunogenicity and protective efficacy. Some of these strategies have already progressed to clinical trials with humans. The study was studied by Vaccine Analytical Research Development and Vaccine Process Development Merck & Co., Inc., Kenilworth, NJ, USA.

Synthetic Long Peptides

FRET peptide helped researchers identify a small molecule drug candidate

LifeTein FRET peptide helped researchers identify a small molecule drug candidate, DMA-135, against Enterovirus 71 (EV71), a virus that causes foot-to-mouth disease with no FDA approved drugs.

The Tat-derived FRET peptide, (5-FAM)-AAARKKRRQRRRAAA-Lys(TAMRA), from LifeTein, binds strongly to the SLII (Stem Loop II) of Enterovirus 71 (EV71) with a dissociation constant Kd of 24.5 +/-4.7 nM. It has been recently used in the fluorescent indicator displacement assay (FID assay) and helped to identify a potential drug candidate, DMS-135, from the small molecule library, capable of internal ribosome entry site (IRES) targeting to block viral replication.

For the FID assay, when the peptide is bound to RNA, FRET is facilitated, allowing for excitation of FAM (485nm) and emission detection from TAMRA (590nm). When the small molecule drug candidate displaces the peptide from the RNA (SLII) secondary structure, the FRET is disabled, and TAMRA is not fluorescent. Such displacement and fluorescence change allow quantification of the binding affinity of the small molecules on the target RNA.

The peptide and its FID assay may prove generally useful in screening and identifying small molecules for viral RNA binding and inhibition.

SARS-CoV-2 (2019-nCoV) vaccine

SARS-CoV-2 (2019-nCoV) peptide vaccine
Covid nasal spray vaccine: synthetic peptides

The SARS-CoV-2 virus (a.k.a. 2019-nCoV; disease: COVID-19) is responsible for the plague year of 2020. The Pfizer-BioNTech and Moderna mRNA vaccines have now been approved for emergency use and more are coming down the pipeline.

The best vaccine should be high safety, low cost, and ease of production and administration. This paper described an interesting citizen-science vaccine based on synthetic peptides.

https://radvac.org/wp-content/uploads/2020/07/White-Paper-SARS-CoV-2-vaccine-ver-2-3-2.pdf

Synthetic peptide synthesis provides the freedom to design epitopes of sufficient length for immunogenic stimulation but is predicted not to trigger these serious side effects. Synthetic peptides are inexpensive and can be made to order quickly. Many simple linear epitopes can be generated without special conformational constraints. The peptide antigens can be delivered by nanoparticles intranasally.

The following peptide sequences were chosen as self-administered vaccines at about 5 to 7 micrograms of each peptide per dose of vaccine.

  1. Spike 436-460, a.k.a. Spike1, NSNNLDSKVGGNYNYLYRLFRKSN
  2. Spike 462-476, KPFERDISTEIYQAd
  3. Spike 478-502, kPCNGVEGFNCYFPLQSYGhQPTNG
  4. Spike 550-574cir, cgLTESNKKFLPFQQgGRDIADTcD
  5. Spike 375cir, cSrdYNSASFSTFKsYGVSPTKcND
  6. Spike 522cir, CGPKKSTNLVKNKsVNFNFNcd
  7. Spike 804-820cir, cILPDPSKPSKRSFcgD
  8. Spike 802-823cir, FSQcLPDPSKPSKRSFcEDLLF
  9. Orf1ab 1544-1564cir (non-circularized), cFHLDGEVITFDNLKTLLSLREct
  10. Spike 462-501, KPeERDgSTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN

Order your testing sample peptides from here: https://www.lifetein.com/peptide-product/peptide-vaccine-testing-samples-p-11049.html

The pivotal role of TMPRSS2 in COVID19

Transmembrane serine protease 2 (TMPRSS2) is a serine protease that in humans is encoded by the TMPRSS2 gene. It is a cell surface protein primarily expressed by endothelial cells across the respiratory and digestive tracts. The protein contains a type II transmembrane domain, a receptor class A domain, a scavenger receptor cysteine-rich domain and a protease domain.

Recent evidence suggested that SARS-CoV-2 uses the ACE2 receptor for cell entry, in synergy with the host’s TMPRSS2. The viral S glycoprotein is cleaved by TMPRSS2, thus facilitating viral activation. As TMPRSS2 is a serine protease, it primes the spike-domain (S) of SARS-CoV-2 by cleaving as the S1/S2 sites. TMPRSS2 activity is crucial for cell entry and viral pathogenesis. In a recent in vitro study by Hoffmann et al., the TMPRSS2 inhibitor camostat mesylate blocked the SARS-CoV-2 entry into primary lung cells, suggesting that TMPRSS2 could represent a potential target in SARS-CoV-2 treatment. This drug is approved for clinical use already in Japan for unrelated illnesses and could serve to be an important therapy for COVID-19.

TMPRSS2 in COVID19
SARS-CoV-2 uses the ACE2 receptor for cell entry, in synergy with the host’s TMPRSS2

Receptor-binding Domains of SARS-CoV-2, LT5578, Cited by Nature

A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2, LT5578, was cited by Nature (Potently neutralizing and protective human antibodies against SARS-CoV-2, volume 584, pages443–449(2020)). This peptide was synthesized in 6 days. This is part of the receptor-binding domain (RBD). It is a critical determinant of virus-receptor interaction and thus of viral host range and tropism. The RBD also includes important viral-neutralizing epitopes (21–23), and it may be sufficient to raise a protective antibody response in inoculated animals.

Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner.

Lately, a recombinant SARS-CoV-2 Spike S (S1+S2) Protein was produced by LifeTein. The amino acid sequences of recombinant protein was derived from (Q14 – Q1208) of accession# YP009724390.1. The SARS-CoV-2 spike (S) protein is composed of two subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell membranes. The S RBD protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity, during infection with SARS-CoV-2 (2019-nCoV) as in recent COVID-19 outbreak.

SARS-CoV-2 receptor binding domain structure
Schematic of the SARS-CoV-2 structure; the illustration of the virus is available at doi: https://doi.org/10.1371/journal.ppat.1008762.g003.

Aureocin A53 is an antimicrobial peptide produced by Staphylococcus aureus A53

LifeTein successfully synthesized Aureocin A53, a highly cationic 51-residue peptide containing ten lysine and five tryptophan residues,   Formyl-MSWLNFLKYIAKYGKKAVSAAWKYKGKVLEWLNVGPTLEWVWQKLKKIAGL, using solid-phase peptide synthesis approach. A53 is a Class II bacteriocins. It was originally isolated from Staphylococcus aureus A53 and is active against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus. The mode of action was proposed as insertion into lipid bilayer and consequent membrane leakage. The solid-phase peptide synthesis provide an inexpensive alternative to isolation from bacteria culture or recombinant expression.

Aureocin A53 Peptide

Aviptadil Fast-Tracked for Respiratory Distress in COVID-19

It was found that RLF-100 (Aviptadil) is associated with rapid respiratory failure recovery among COVID-19 Patients. The clinical findings may be based on evidence that VIP inhibits the replication of the SARS-CoV-2 virus in human lung cells and immune cells (monocytes). No other antiviral agent has demonstrated rapid recovery from viral infection and demonstrated laboratory inhibition of viral replication. It is a patented formulation of aviptadil (synthetic human Vasoactive Intestinal Polypeptide, VIP), which has been granted FDA fast track designation, FDA emergency use IND authorization, and an expanded access protocol. Aviptadil is an injectable formulation of the vasoactive intestinal polypeptide (VIP) in combination with the adrenergic drug phentolamine.

Aviptadil (Senatek), Vasoactive intestinal polypeptide

Peptide Library: SARS-CoV-2 Receptor Binding Domains

Coronavirus
Coronavirus receptor binding domain. The key receptor binding domain (residues 319-541) is highlighted in yellow. Variable amino acid residues between SARS-CoV-2 and SARS-CoV are highlighted in cyan. Tyr 489, Asn 487, Gln 493, Tyr 505 are important for ACE2 binding.

LifeTein can help in your research with custom peptide synthesis of the following specific proteins: SARS-CoV-2 Receptor Binding Domains, SARS-CoV-2 Nucleocapsid Fragments, T-cell and B-cell Epitopes of SARS-CoV-2, Fusion Inhibitors Targeting HR1 Domain of the SARS-CoV-2 Spike Proteins, Inhibitors of SARS-CoV-2 Mpro/3CLpro/C30 Endopeptidase, ACE2 Inhibitors and Substrates, and AT2 Receptor Agonists and Antagonists.

Pool of 22 peptides derived from a peptide design (15mers with 5 aa overlap) through the receptor binding domain of S1 protein.

Modifications: N-Terminal: Biotin Labeling

Amount: 1mg per peptide

Purity: 95%

Delivery Format: Freeze dried powder

Application(s): Antibody screening, T-cell assays, Immune monitoring, Antigen specific T-cell stimulation, Cellular immune response

Indication(s)/Topic(s): Covid-19, Infection, Respiratory infection

Delivery Time: 2 weeks

SARS-CoV-2 Receptor Binding Domains Overlapping Peptide Pools:

  • QPTESIVRFPNITNL
  • NITNLCPFGEVFNAT
  • VFNATRFASVYAWNR
  • YAWNRKRISNCVADY
  • CVADYSVLYNSASFS
  • SASFSTFKCYGVSPT
  • GVSPTKLNDLCFTNV
  • CFTNVYADSFVIRGD
  • VIRGDEVRQIAPGQT
  • APGQTGKIADYNYKL
  • YNYKLPDDFTGCVIA
  • GCVIAWNSNNLDSKV
  • LDSKVGGNYNYLYRL
  • YLYRLFRKSNLKPFE
  • LKPFERDISTEIYQA
  • EIYQAGSTPCNGVEG
  • NGVEGFNCYFPLQSY
  • PLQSYGFQPTNGVGY
  • NGVGYQPYRVVVLSF
  • VVLSFELLHAPATVC
  • PATVCGPKKSTNLVK
  • TNLVKNKCVNFNFNG

The Cationic Host Defense Peptides Could Be Used To Kill Enveloped Novel Coronavirus SARS-CoV-2

Direct antimicrobial mechanisms of cationic host defense peptides

The cationic host defense peptides (CHDP), also known as antimicrobial peptides, could be used to kill enveloped viruses such as the 2019 Novel Coronavirus SARS-CoV-2. The peptides have the potential to destabilize the viral envelope on contact, damaging the virions and inhibiting infectivity. The specific antiviral peptide may bind to cellular receptors involved in viral infection or peptide-mediated aggregation of viral particles. The antiviral peptides could create an ‘antiviral shield’ at mucosal surfaces and prevent replication and spread of the Coronavirus if upregulated after the initial infection.

During pandemics, where there is insufficient time to produce vaccines (such as the outbreak of respiratory illness Covid-19 first detected in Wuhan, China), the cationic host defense peptides could be the first-line antiviral treatments.

Some of the antimicrobial peptides are the human cathelicidin LL-37 and β-defensins. Cathelicidins are immunomodulatory antimicrobials with an important role in the regulation of the inflammatory response. The only human cathelicidin, LL-37, is the most well-studied peptide in this family. LL-37 is an α-helical peptide. While defensins have a common β-sheet core stabilized with three disulfide bridges between six conserved cysteine residues.

Direct antimicrobial mechanisms of cationic host defense peptides can be mediated by membrane translocation of the peptides followed by binding to intracellular targets such as nucleic acids and/or proteins to kill bacteria. Proline-rich antimicrobial peptides use inner membrane transporters as Trojan horses to gain entry and bind to intracellular targets such as nucleic acids or nascent proteins. And subsequently affect cell processes such as replication, transcription, translation, protein folding, and cell wall synthesis.

At this stage, only a few peptide-derived treatments have made it to market such as PAC-113, a histatin analog, and dalbavancin, a semisynthetic lipoglycopeptide.

Despite the limited understanding of structure-function relationships, the potential of peptide-based therapies remains a promising new clinical direction for the Coronavirus.