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.
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 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.
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
A simple protocol: Click chemistry of antibody-DNA conjugation
- 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
- Simon et al. (2012) Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries. Bioconjugate Chem. 23(2):279.
- 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.
- Campbell-Verduyn et al. (2011). Strain-Promoted Copper-Free Click Chemistry for 18F Radiolabeling of Bombesin. Angew. Chem. Int. Ed. 50:11117.
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 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.
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
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
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
Reference: M3 peptide was purchased from LifeTein.
Cellular imaging by targeted assembly of hot-spot SERS and photoacoustic nanoprobes using split-fluorescent protein scaffolds
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.
- 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
Glucagon-Like Peptide 1, GLP – 1 (7 – 36), amide, human:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR – NH2
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.
PTH 1-34: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF
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
Zonula occludens toxin (Zot) and its biologically active fragment, delta G, have been shown to reversibly open tight junctions (TJ) in endothelial and epithelial cells. AT1002, a six-mer synthetic peptide H-FCIGRL-OH of ZO toxin was identified and synthesized that retains the Zot permeating effect on intercellular TJ. It was found that AT1002 disrupts the epithelial barrier while larazotide acetate restores barrier function by rearrangement of actin. In addition, AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. So this synthetic peptide AT1002 is a tight junction modulator with promising permeation-enhancing activity.
The C-terminal amidated AT1002 FCIGRL-NH2 showed enhanced nasal drug delivery and may lead to the development of a practical drug delivery technology for drugs with low bioavailability.
The synthetic peptide AT1002 was synthesized by LifeTein.
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