Trans-activator of transcription (Tat) with nuclear translocation signal peptide drug candidate

Endocytosis TAT Peptide

Endocytosis TAT Peptide

Nuclear translocation of annexin A1 (ANXA1) has been reported to participate in diverse cellular processes. It was found that the amino-acid residues from R228 to F237 function as a unique nuclear translocation signal (NTS) and are required for nuclear translocation of ANXA1.

Trans-activator of transcription (Tat) is a cell-penetrating peptide. The peptide can translocate numerous proteins, peptides, DNA, RNA, and small drugs into the cytoplasm with high efficiency. The Tat-NTS (NH2-YGRKKRRQRRR-RSFPHLRRVF-CONH2) peptide was found to specifically block the interaction of ANXA1 with importin β. This blocking would inhibit the nuclear translocation of ANXA1, consequently protecting neurons from ischemic stroke damage. So the Tat-NTS peptide can be used as a novel and potentially promising new therapeutic candidate for the treatment of ischemic stroke.

From the experiment, the Tat-NTS peptide-treated mice displayed remarkable cognitive improvement. In addition, the Tat-NTS peptide had no obvious effect on neuronal apoptosis. So the administration of Tat-NTS peptide to dissociation of ANXA1–importin β interaction may be a new drug in ischemic stroke therapy.

The Tat sequence is originally from a crucial non-structural protein of human immunodeficiency virus (HIV). The function of Tat is to bind to the viral long terminal repeat (LTR) and activate cellular transcription machinery to initiate transcription of the viral proteins. Later, it was found that this 11-amino acid positively charged peptide can pull diverse molecules across cell membranes in vitro and in vivo. Fusion proteins constructed with TAT rapidly enter and exit cells and cross intracellular membranes. Electrostatic interactions between TAT and the cell membrane have been implicated as a part of the mechanism of transduction. Neither apoptosis nor necrosis is induced in cells after exposure to TAT.

Molecular grafting of macrocyclic disulfide-rich peptides

Bioactive peptides have potential as drug leads. However, one of the peptides’ limitations is their poor stability.

One solution is to graft peptides onto suitable molecular scaffolds. When the peptide scaffolds are rich in disulfide bonds and have no free ends for proteases to attack, the peptide drug would be a stable candidate.

Cyclic disulfide-rich peptides: Stability Matters
Many natural peptides have stable and conserved cyclization. The cyclic chlorotoxin can preferentially bind to tumor cells. The defensin, an antimicrobial peptide, is an 18 amino acid peptide with three disulfide bonds in a laddered arrangement. The cyclotide has about 30 amino acids with three disulfide bonds in a knotted configuration. The cyclic conotoxin cVc1.1 has potent analgesic activity.

disulfide rich cyclic natural product

disulfide-rich cyclic natural product

Molecular grafting
The purpose of molecular grafting is to make a linear peptide sequence into a disulfide-rich peptide with desired stability or oral bioavailability. It was found that the stability of linear peptide epitopes in human serum can be enhanced by grafting them onto a stable scaffold. For example, a crafted peptide comprising an epitope from myelin oligodendrocyte glycoprotein (MOG) is resistant to degradation in human serum (>24 h) and remained intact in strong acid after 24 hours. In the MOG study, the epitope was grafted into the β-turn of Kalata B1 to mimic its native conformation and it turned out to have the increased bioactivity and improved affinity and receptor selectivity. A grafted peptide based on the cell-penetrating peptide cyclotide MCoTI-I demonstrated the delivery of the bioactive peptide across the cell membrane and into the intracellular space.

Cyclotide Peptide Synthesis

Cyclotide Peptide Synthesis

How to make molecular grafting
1. Selection of a suitable epitope and scaffold pair. The termini of the epitope should not be essential for its activity. The size of the epitope is an important consideration. Epitopes ranging from 10 amino acids to 20 amino acids are frequently used. Here are a few examples of the epitope size from the disulfide-rich peptide scaffolds by chemical design: cyclic conotoxin (6 amino acids), defensin (6, 7, or 12 amino acids), Kalata B1 (3, 6, or 9 amino acids), McoTI (3, 7, 9, 16, 18, or 21 amino acids), Cyclic chlorotoxin (10 amino acids).

2.Structural and functional characterization.
The peptide epitope should be from the fragments of an interacting protein or functional and bioactive known domains from the screening assays such as a phage display library.

Then where should the epitope be grafted onto the scaffold?There are a few possible suggestions: insertion between two existing residues; substitution of one or more residues in a single loop; replacement of residues that span across connected loops; or replacement of most of the native residues of the scaffold.

3. How to make the grafted peptides?
The grafted peptides have been made using solid-phase chemical peptide synthesis. First, the reduced linear precursors are assembled. They are the sequences from the scaffold and epitope. Then oxidization and cyclization are performed to form the final product. However, synthesis of more complex peptides with multiple disulfide bonds can be challenging. A peptide comprising just three disulfide bonds would have 15 different connectivities. In addition, grafting of an epitope onto a scaffold may not be folded correctly, or it does not have the desired activity.

Future perspectives
It is encouraging that many grafted peptides have exhibited oral activity. The cyclic disulfide-rich scaffolds have enhanced stability. However, not all grafted peptides fold into the desired conformation. So a more detailed understanding of how grafting affects folding of disulfide-rich peptides would be beneficial.

disulfide formation

disulfide formation

Simple method to prepare antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates

We describe a simple method for preparing antibody-peptide, antibody-oligonucleotide or antibody-compound conjugates and discuss its applications in drug delivery and new drug design. Conjugation is based on alkyne-azide cycloaddition. This Cu-free click reaction starts from the dibenzocyclooctyne (DBCO) moiety-activated antibodies and subsequently linked covalently with an azide-modified peptide, oligonucleotide or compounds. The reaction is performed under physiological conditions and has no adverse effects on antibodies or proteins. This can also be used as the click chemistry fluorescence labeling and the click chemistry in peptide-based drug design.

However, the copper-catalyzed alkyne-azide cycloaddition (CuAAC) is not suitable for applications involving functional biomolecules because copper ions can cause protein denaturation.

Measuring the protein levels directly is challenging. However, the signals can be amplified by immuno-PCR using oligonucleotide-attached antibodies to detect protein indirectly.



1. Conjugation of DBCO to the Antibody. The DBCO-PEG5-NHS was used to react with the NH2 groups on the antibody. The inclusion of a PEG5 linker improves the water solubility of the hydrophobic DBCO, introduces a spacer and flexibility between the antibody molecule and the peptide/oligonucleotide or compounds. This will alleviate the steric effect of the antibody on the enzymatic reactions.

2. Prepare the azido-Peptide or azido-oligonucleotide. LifeTein provides click chemistry modified peptide synthesis: N-terminal azide-peptide/oligo or C-terminal peptide/oligo-azide.

3. Covalent attachment of the peptide/oligonucleotide to the antibody. The reaction between DBCO and azide is slow compared to CuAAC reaction. The reaction time of 16–18 h in PBS at 4 °C is ideal to increase the final product yield. The DBCO-antibody in the intermediate reaction is stable.

Copper-Free Click Chemistry Antibody-DNA Conjugation

Typically, there are three biological functional groups on the peptide for the further conjugation: amino ( –NH2 ), carboxyl ( –COOH), and thiol ( –SH). The most effective way is to utilize the free thiol groups from cysteine. The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules.

The click chemistry is another efficient method to conjugate the peptide with other biomolecules. The peptide can be modified with azide groups (–N3). The novel Copper-free Click Chemistry is based on the reaction of a diarylcyclooctyne moiety (DBCO) with an azide-peptide reaction partner. This click reaction is very fast at room temperature and does not require a cytotoxic Cu(I) catalyst, resulting in almost quantitative yields of stable triazoles. The DBCO allows Copper-free Click Chemistry to be done with live cells, whole organisms, and non-living samples. Within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls, which are naturally present in many biomolecules. The reaction of the DBCO group with the azide group is significantly faster than with the sulfhydryl group (–SH, thiol).

One example of the peptide drug conjugations is the antibody-biomolecule conjugate.

click chemistry: DBCO-azide

click chemistry: DBCO-azide

A simple protocol: Click chemistry of antibody-DNA conjugation

Pre-conjugation considerations

  • Remove all additives from antibody solutions using dialysis or desalting.
  • Remove BSA and gelatin from antibody solutions.
  • Concentrate the antibody after dialysis or purification.

Activation of antibodies with DBCO-NHS ester

  • Mix antibody with 20-30 fold molar excess over antibody of DBCO-NHS ester dissolved in DMSO.
  • Incubates at room temperature for 30 min or 2 hours on ice.

Quenching activation reaction

  • Add Tis-Hcl (50-100mM, pH 8) to the reaction.
  • Incubate at RT for 5 min or 15 minutes on ice.

Equilibration and removal of non-reactive DBCO-NHS ester by Zeba column (Follow the manufacturer’s instruction)

Copper-Free click reaction

  • Mix DBCO-NHS ester labeled antibody with 2-4 times molar excess of azide-modified Oligos.
  • Incubated overnight (around 10-12 hours) at 4°C or 3-4 hours at room temperature.

Validation of conjugation and purification by HPLC

Selected References:

  1. Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
  2. Arumugam et al. (2011). [18F]Azadibenzocyclooctyne ([18F]ADIBO): A biocompatible radioactive labeling synthon for peptides using catalyst-free [3+2] cycloaddition. Bioorg. Med. Chem. Lett. 21:6987.
  3. Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.

A simple protocol: Maleimide labeling of peptide and other thiolated biomolecules

The reaction of maleimides with thiols is widely used for bioconjugation and labeling of biomolecules such as proteins and peptides. Maleimides are electrophilic compounds which show high selectivity towards thiols.
1. Dissolve the peptide or other biomolecules containing thiol in degassed buffer (PBS, Tris, or HEPES) at pH 7-7.5.
2. Add a 100x molar excess of TCEP (tris-carboxyethyl phosphine) reagent to reduce disulfide bonds.
3. Dissolve maleimide in DMSO or fresh DMF (1-10mg in 100uL).
4. Add dye solution such as cy5 maleimide to thiol solution (20x fold excess of dye), flush with an inert gas, and close tightly.
5. Mix thoroughly and keep at room temperature or 4C overnight.
6. Purify by gel filtration, HPLC, FPLC, or electrophoresis.

Personalized treatment using synthetic peptides

personalized medicine using synthetic peptides

personalized medicine using synthetic peptides

Interest in personalized treatment has been fuelled by the concept to tailor therapy with the best response and highest safety margin to ensure better patient care. Personalized medicine holds promise for improving health care while also lowering costs.

An immunogenic personal neoantigen vaccine for melanoma patients using the synthetic peptides provides an opportunity to develop agents that are targeted to patient groups that do not respond to medications as intended and for whom the traditional health systems have otherwise failed.

The T cell epitopes with tumor-specific expression arising from non-silent somatic mutations are not expressed in normal tissues. These neoantigens are mutated peptides with the high-affinity binding of autologous HLA molecules.

The vaccination with neoantigens can induce new T cell specificities in cancer patients. Using the synthetic peptides as a personalized vaccine, researchers found that of 6 vaccinated patients, 4 had no recurrence at 25 months post-vaccination.

The T cells discriminated mutated from wildtype peptide antigens, and directly recognized autologous tumor. From this study, immunizing peptides were selected based on HLA binding predictions. Each patient received up to 20 long peptides in 4 pools.

Long peptide synthesis by click chemistry

Some fusion protein or chimeric proteins could never be produced from the e.coli expression system, especially when several hydrophobic sequences are involved in the functional domains. Obtaining peptides sized 100–200 amino acids using chemical synthesis is much faster and cheaper than cloning and overexpressing in Escherichia coli. In addition, the resulting peptide is always correct. Chemical synthesis can be used to incorporate non-genetically encoded structures, such as D-amino acids, into the protein in a completely regular fashion. Synthetic peptides eliminate problems such as poor or no expression, cloning errors, tags like FLAG or 6-His, or the mistranslation of non-preferred codons in prokaryotic hosts. Artificial amino acids that have isosteric side chains can be used to investigate the functional importance of specific residues. All these chimeric proteins can be achieved by the peptide design and synthesis using the click chemistry.

Long peptide synthesis by click chemistry

Long peptide synthesis by click chemistry

Post-translational modifications: Methylated peptides

Post-translational modifications of histone proteins, such as acetylation, methylation, and phosphorylation, play essential roles in regulating chromatin dynamics. The mono-, di-, or tri-methylated peptides can be used to study the protein-protein interactions. The peptide methylation occurs at arginine or lysine residues, resulting in methyl-arginine or methyl-lysine. In a new study, an H3 histone tail mimicking peptides were used to bind with the ASHHH2 CW domain.The monomethylated ARTK(me1)QTARY, dimethylated ARTK(me2)QTARY, and trimethylated ART- K(me3)QTARY were synthesized by LifeTein
(95% purity by mass spectrometry).

The methylated peptide is an important tool to study the histone methylation. Histone methylation can be associated with either transcriptional repression or activation. There is an emerging realization that DNA and histone lysine methylation in mammals are highly interrelated. Targeting of DNA methylation is mechanistically linked to H3K9 methylation. For example, the p53 gene is the most frequently mutated tumor suppressor gene in human cancers. Upon genotoxic stresses, p53 proteins are activated in the setting of multiple post-translational modifications such as phosphorylation, methylation and acetylation for full activation. The arginine methylation includes Arg(Me), Arg(Me)2 asymmetrical or Arg(Me)2 symmetrical.

Post-translational modifications of p53

Post-translational modifications of p53


Noble metal gold and silver nanoparticle are conjugated with peptides for cellular imaging

Noble metal gold (Au) and silver (Ag) nanoparticle (NPs) are used to conjugate with M3 peptides. The AuNPs-sGFP andAuNPs-M3 peptide form SERS active hot spot through self-assembly and GFP complementation. The nanoparticles self-assemble into surface-enhanced Raman-scattering (SERS) nanoclusters. The nanocluster can be used as contrast agents for multimodal SERS and photoacoustic microscopy with single-cell sensitivity.

AuNPs coated with M3 peptides-GFP

AuNPs coated with M3 peptides-GFP

Reference: M3 peptide was purchased from LifeTein.

Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds

How to generate highly stable D-amino acid analogs of bioactive helical peptides?

Using D-amino acids as the building blocks for bioactive peptides can dramatically increase their potency. In this study, the authors generated a database of ∼2.8 million D-peptides using a mirror image of every structure in the Protein Data Bank (PDB). The critical or hotspot residues were studied. Residues critical to target binding and activity can then be ideally done experimentally such as alanine scanning mutagenesis. It can also be carried out computationally such as thermodynamic integration or free energy perturbation.

Two peptides were tested to prove the concept: GLP-1 and Parathyroid Hormone. Both (L)- and (D)-peptides were synthesized by Lifetein LLC.

  1. GLP-1 is a helical GPCR agonist as a diabetes mellitus and obesity treatment. Hotspot and junction residues are annotated in green and blue, respectively. The authors investigated the ability of (D)-GLP1 peptide to induce activation of GLP1R and compared the response with native (L)-GLP1 peptide. It was found that the D-GLP-1 performed well and resistance to protease degradation. The retro-inversion (RI) reversing the (D)-peptide sequence was used in the experiment.
D amino acid peptides

D amino acid peptides

Glucagon-Like Peptide 1, GLP – 1 (7 – 36), amide, human: 

2.  Parathyroid Hormone (PTH) is an FDA-approved treatment for osteoporosis. The (D)-PTH activates PTH1R with a potency and efficacy comparable to (L)-PTH. And more than 85% of the (D)-PTH analog is still detectable at six hours.


Conclusion: The D-Protein Data Bank (PDB) can be used to search and find therapeutically active topologies. The D-PDB could be a key tool for finding stable lead molecules in early-stage drug discovery.

Hot spot residues for receptor binding

Hot spot residues for receptor binding


Method to generate highly stable D-amino acid analogs of bioactive helical peptides using a mirror image of the entire PDB