Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as necessary co-receptors to activate FGF receptor (FGFR) signaling.
A series of peptides were synthesized by LifeTein and used for the competition binding assay. Both the KL1 and KL2 domains of β-Klotho participates in ligand interaction. The FGF19 peptide was used for alanine scanning mutagenesis. It was found that a single amino acid mutations in either region were sufficient to abolish β-Klotho binding. FGF19 and FGF21 function through β-Klotho to regulate glucose and lipid metabolism.
How to perform the solid-phase binding assay
1. The 96-well plates were coated overnight at 4 °C with 2 µg/mL of antibody in PBS.
2. Plates were washed twice with PBST and blocked with 3% (w/v) BSA in PBS for 1.5 hours at room temperature.
3. The conditioned media containing β-Klotho were added to the plates and incubated for 1.5 hours at room temperature.
4. Plates were washed a few times.
5. The peptide mutation FGF21 and an anti-β-Klotho antibody were biotinylated with EZ-Link Sulfo-NHS-LC-Biotin at the indicated concentrations.
6. After wash, streptavidin-HRP was used for detection.
7. EC50 values were determined.
How to do competition binding assay?
1. The WT and mutant peptides were custom synthesized and purified (>95% purity) by LifeTein.
2. Binding of FGF19 and FGF21 peptides to β-Klotho was assessed.
3. The β-Klotho ECD 6 × His, varying amounts of FGF19 and 21 peptides, and biotinylated human FGF19 or FGF21 protein were prepared.
4. The streptavidin donor beads and nickel chelate acceptor beads were added to the plates.
5.Plates were incubated for 3 hours at room temperature protected from light and read on the Plate Reader.
The folate receptor alpha (FRα) is highly expressed in ovarian cancer and not in normal tissues. An FRα binding peptide C7 (Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser, MHTAPGWGYRLS) was found to bind to FRα expressing cells. This tumor-targeting peptide was proved by both phage homing experiment and fluorescence imaging.
Tumor Targeting of Conjugated Synthetic Peptides
1. The FITC-conjugated peptide FITC-MHTAPGWGYRLS was dissolved in PBS.
2. The peptide was injected intravenously into a tumor-bearing nude mouse.
3. After 2 h, the tumor and other organ tissues were harvested and analyzed using a fluorescence imaging system.
Cell internalization of Synthesized Peptide
1. Cells were seeded in 24 well plates containing coverslips and incubated for 24 hours in a medium with FBS.
2. FITC conjugated peptide was incubated with the cells for 4 hours at 37 °C.
3. The cells were washed once with PBS and fixed in 4% paraformaldehyde.
4. Cells were washed three times with PBS and stained with DAPI for 20 min at room temperature.
5. Internalized fluorescent signals were imaged with a confocal microscope.
This tumor-specific peptide could be a potent and selective ligand for FRα. It has a great potential for the delivery of cancer therapeutics or imaging agents to express tumors.
Cancer is expected to surpass the current number one, cardiovascular diseases by 2030 as the leading cause of death.
The targeting peptides can be considered as an alternative vehicle for the delivery of anti-cancer drugs because of their lower molecular weight and excellent tolerability by human bodies.
The following modification can prolong the half-life of the peptides from the degradation by blood proteases: forming cyclization within a peptide, blocking of the C- and N- terminus, replacement of standard L-type amino acids by their D-amino acid counterparts, using unnatural amino acids incompatible with endogenous proteases.
About targeting peptides:
1. Somatostatin (SST) derivatives: Binding of the natural ligand somatostatin peptide to the receptors leads to inhibition of overexpressed SSTR2 and 5 in breast cancer. For example, cyclic SSTR agonist octreotide (fCfDWKTCT), selectively binds SSTR2 and 5.
2. Peptide-derivatives of gastrin-releasing peptide (GRP)
The gastrin-releasing peptide receptor is associated with the prostate and breast cancer. It was found that bombesin (YQRLGNQWAVGHLM) and its derivatives could be used as targeting peptides for the detection of prostate cancer via PET and CT screening.
3. Peptides targeting tumor microenvironment
The glycoprotein prosaposin (PSAP) can inhibit metastases from breast and lung cancer in preclinical models. A cyclic PSAP peptide (DWLPK) could hinder metastatic spread and restrain tumor development. The synthetic antagonist of CXCR4 called NT21MP (LGASWHRPDKCCLGYQKRPLP) exhibits anti-tumor activities through decreased adhesion and migration of breast cancer cells. The peptide R (RACRFFC) targeting of CXCR4, showed capacities to remodel the tumor stroma.
4. Peptides targeting the tumor pH and temperature
The pHLIP (pH-Low Insertion Peptide), ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET, can be inserted into cell membranes as an a-helix under low pH conditions. The pHLIP peptide increases the uptake of the peptide-coated drug to tumors compared to the naked particles. Thus, the local tumor microenvironment can be used to trigger peptide drug formulations to respond accordingly.
The elastin-like polypeptides (ELP) are conjugated to the cell-penetrating peptide Bac (RRIRPRPPRLPRPRPRPLPFPRPG) for improved cellular penetration to deliver gemcitabine for pancreatic cancer.
5. Peptides targeting tumor tissues
A widely used endothelial-binding peptide is The tripeptide arginine-glycine-aspartic acid (RGD), an endothelial-binding peptide, that has high specificity towards integrins for anti-tumor and anti-angiogenic treatments.
The radiolabeled, PEGylated RGD has been used as a PET probe to detect gliomas, and the iron-oxide nanoparticles coupled RGD was used for the MR imaging of brain tumors.
The cyclic iRGD (CRGDKGPDC) is a prototypic tumor-penetrating peptide binding integrins. The iRGD peptide increased tumor tissue penetration and the delivery of drugs, nanoparticles, or antibodies in vivo.
The cRGD (RGDdYK) and cilengitide (cRGDf [N-Me]V) with the combination therapy of temozolomide (TMZ) radiochemotherapy were used in the clinical trials.
A linear targeting peptide called CooP (CGLSGLGVA) binds the mammary-derived growth inhibitor (MDGI). The MDGI is a fatty acid-binding protein that is highly expressed at the cell membrane of malignant glioma cells.
The coop peptide is a homing peptide targeting glioma cells and tumor-associated blood vessels. The chemotherapeutic drug conjugated CooP peptide can reduce the number of invasive tumor cells.
The cell penetration peptides are the cure for difficult-to-access cancers such as brain tumors. This endothelial-specific peptide with enhanced penetrance would allow better passage of the drug conjugates through the blood-brain barrier.
The peptide drugs have the benefits of high specificity, low antigenicity, low cost, and simple production. The peptides have the potential for the development of therapy options for various tumors in the field of personalized medicine of cancer.
