FLUORESCENT PEPTIDES: VALUABLE TOOLS FOR MEDICAL RESEARCH

Imaging of cellular structures and the understanding of molecule-molecule interactions have utmost impact on the advances in modern medicine. Thereby, fluorescently labeled peptides are indispensable tools for a number of methods in cutting-edge research.

Fluorophores

Fluorophores absorb light in the UV or visible range and re-emit part of the energy as radiation (fluorescence). The emitted radiation has a longer wavelength, than the excitation light (Stokes shift). The emission wavelength furthermore does not depend on the wavelength of the excitation light. Fluorescent peptides are utilized for (confocal) fluorescence microscopy or protease activity assays. For example, peptide substrates with amino-terminal fluorophores and carboxy-terminal biotin moieties are used. After proteolytic cleavage, the uncut peptides and carboxy-terminal cleavage products are removed using streptavidin. The remaining fluorescence provides a measurement for the proteolytic activity of the enzyme. For fluorescence microscopy, the fluorophores should have a high stability against photo-bleaching under the high light intensities applied. In general, the fluorophores should exhibit a strong fluorescence.

FRET

In fluorescence (or Förster) resonance energy transfer (FRET), the fluorescence of an excited fluorophore (donor) is absorbed by a second dye label (acceptor). Donor and acceptor are also often referred to as “FRET-pair”. In contrast to collisional or dynamic quenching, a direct contact between fluorophore and quencher is not required. Since FRET typically takes place in a distal range between 1-10 nm, it can be employed to measure processes on a molecular scale. The acceptor can either act as a secondary fluorophore (ratiometric FRET), or, can abolish (quench) the donor fluorescence, the latter used for example in protease assays (Figure 1). In order to obtain an efficient FRET, the emission spectrum of the donor and the absorption spectrum of the acceptor label should overlap as complete as possible.

Table 1: Selection of Fluorescent Peptides in Development for Imaging

Product Candidates

BLZ100 is a tumor paint being developed by Blaze Bioscience for use as an imaging agent for cancer surgery involving a range of cancer types including brain, breast, prostate, lung, colorectal, skin and sarcomas. The company aims to provide surgeons with the ability to see cancer cells at a high resolution throughout surgery and thereby provide better detection and allow for the precise removal of cancer. The tumor paint consists of chlorotoxin, a tumor penetrating peptide, conjugated to indocyanine green dye. In April 2016, Blaze completed a Phase I study of BLZ100 in adult subjects with glioma undergoing surgery (1). Blaze announced interim clinical data from this Phase I study in November 2015 and the data suggested that BLZ100 is well tolerated and tumor-specific fluorescence can be achieved in vivo. The product is currently in multiple Phase I proof-of-concept clinical studies (2).

Avelas Biosciences is developing AVB620 as an in vivo surgical diagnostic agent for breast cancer. AVB620 is a protease-activated peptide that detects, marks, and diagnoses cancer. AVB620 is administered prior to surgery and the imaging is performed with a fluorescence imaging camera system. The technology is designed to enable surgeons to identify critical cancer margins and stage lymph nodes (3). In April 2015, Avelas announced that it initiated a Phase Ib study of AVB620 in women with breast cancer. In May 2016, Avelas reported interim results from the Phase Ib trial of AVB620. The interim analysis showed no toxicity issues and differentiated fluorescence between cancer and non-cancerous tissue. In addition, a preferred dose was identified to take forward into an expansion phase of the trial (1).

Researchers at the University of Michigan are developing KCC peptide for use during colonoscopy procedures. KCC peptide contains 7 amino acids and is attached to FITC (Fluorescein isothiocyanate). The team’s technique involves spraying the fluorescent peptide onto the colon during colonoscopy so that the peptide will illuminate any abnormal or pre-cancerous areas when a special light is used in the scope. KCC peptide is currently in a Phase Ib study to determine if the peptide glows sufficiently (4).

Stanford scientists have designed an integrin-binding knottin peptide, EETI 2.5F, for the imaging of medulloblastomas, the most common type of childhood brain tumors. The knottin peptide is conjugated to a fluorophore and when injected, the peptide attaches to the integrin receptors of brain tumors while the peptide is expelled from healthy tissues. The team’s knottin peptide has been used in mice to image medulloblastomas and the next step is to translate the results to human patients (5).

Researchers at the University of California San Diego are developing a fluorescent nerve binding peptide called F-NP41 that highlights nerves during surgery. This fluorescent probe consists of a 17 amino acid nerve binding peptide coupled to FAM (5(6)-carboxyfluorescein). In a study with mice undergoing surgery, the researchers found that the fluorescently labeled peptide delineated all peripheral nerves within two hours of injection. Potential applications of this fluorescent peptide probe are to prevent accidental transection during surgery or to help with the identification and surgical repair of injured nerves after trauma (6).

Conclusion

Fluorescently labeled peptides are powerful tools that have the potential to positively impact image-guided surgery and diagnostics in the area of oncology. To support the development of fluorescent peptides for research and clinical applications, Bachem provides a one-stop shop for the labeling of peptides and amino acids. We offer peptides and amino acids tagged with Tide Fluor^TM, fluorescent dyes with fluorescent emissions that span the full visible and near infrared spectrum. Tide Fluor dyes are optimized/designed to meet the highest demands in fluorescent intensity and photo-stability and outperform most conventional and proprietary dyes. Please contact our Custom Synthesis department to request a quote for your labeled peptide or amino acid.

References:

1. Medtrack. [Online] [Cited: Jun 9, 2016.]

2. Clinical Trials. Blaze Bioscience. [Online] 2016. [Cited: Jun 9, 2016.] http://www.blazebioscience.com/for-patients.html

3. AVB-620. Avelas Biosciences. [Online] [Cited: Jun 10, 2016.] http://www.avelasbio.com/research-development/avb-620/

4. Study of KCC Peptide Application in the Colon (KCC 1B). ClinicalTrials.gov. [Online] May 10, 2016. [Cited: Jun 10, 2016.]https://clinicaltrials.gov/ct2/show/NCT02156557?term=kcc+peptide&rank=1

5. Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma. Moore SJ, Hayden Gephart MG, Bergen JM, Su YS, Rayburn H, Scott MP, Cochran JR. 36, Sep 3, 2013, Proc Natl Acad Sci U S A, Vol. 110, pp. 14598-14603.

6. Fluorescent peptides highlight peripheral nerves during surgery in mice. Whitney MA, Crisp JL, Nguyen LT, Friedman B, Gross LA, Steinbach P, Tsien RY, Nguyen QT. 4, April 2011, Nat Biotechnol, Vol. 29, pp. 352-356.