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PEPTIDE TRENDS JULY 2016

BACHEM APP – NOW AVAILABLE!

Our new app is now available. Mobile apps present clear advantages concerning customer-facing image. It is possible to achieve greater brand loyalty through attractive videos and virtual representations. The app features news & highlights, multimedia information, a peptide calculator, a peptide game and many more. Explore the new app and benefit from easily accessible, intelligible, durable and objective information including regularly updates.

 

To find out more please click here.

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.

 

Figure 1: Typical protease activity assay using a FRET substrate. Substrate cleavage separates fluorophore (F) and quencher (Q), thus recovering the donor fluorescence.

 

 

Fluorescent peptide labeling

Fluorescent labels (dyes) can be covalently bound to the amino-terminus of the peptides during solid phase synthesis, or, linked site-specifically to already existing peptides. Amine- or thiol-groups in the side-chains of the amino-acids can be used for the labeling, if this does not impair the biological function. Biotinylated peptides can be labeled in situ using fluorescent streptavidin- or avidin-conjugates. Carboxy-terminal labeling is often more difficult, and the feasibility should be clarified in advance.

 

Examples for the use of fluorescent peptides in medical research

In Alzheimer`s Disease research, understanding of the formation of senile plaques is of central importance. A number of fluorescent Aβ peptides are available, representing the main research targets Aβ (1-42) and Aβ (1-40), but also a variety of truncated forms. The studies include for example phagocytosis events in microglia, cell-to-cell transfer of Aβ (1-42), the interaction of Aβ (1-40) with lipid bilayers and the investigation of conformational changes.

 

The enzymes β- and γ-secretase take part in the processing of the amyloid precursor protein (APP) to the mature amyloid β peptide. FRET-labeled substrate peptides containing the wild type or double-mutated (“Swedish”) cleavage site are used for activity assays; the Swedish mutation strongly influences the aggregation behavior of the amyloid β peptides. Further information can be obtained from our e-brochure Amyloid Peptides, Secretase Substrates Inhibitors and FRET Substrates.

 

Matrix-metalloproteases (MMPs) are important target enzymes for the understanding or inflammation, degradation of bone, autoimmune diseases, and cancer. FRET-labeled substrates are available, which usually can be hydrolyzed by several MMPs, but with largely different catalytic efficiency. For additional information please refer to our e-brochure Matrix-Metalloproteinases.

 

 

Faulty regulation in the programmed cell death (apoptosis) may result in a number of severe diseases, and the activation of caspases (cysteine dependent aspartate specific protease) represents a point of no return in the regulatory cascade. FRET-labeled caspase substrates are used for example for the development of new therapeutics for cancer.

 

Tide Fluor and Tide Quencher™ 

We recently included Tide Fluor™ and Tide Quencher™ into our offer of dye labeled peptides. Tide Fluor™ fluorophores outperform most classic and also many proprietary dyes in fluorescence intensity and photo-stability. In combination with Tide Quencher™ acceptors, they are also excellent dyes for FRET-analysis.

 

 

 

 

 

Conclusions

Fluorescently labeled peptides are versatile and valuable tools for medical and biological research. Peptides can be labeled during or after synthesis. Important properties of the fluorophores are a strong fluorescence and, for fluorescence microscopy, a high photo-stability. For FRET-experiments, the emission spectra of the donor and the excitation spectra of the acceptor should overlap as good as possible, which should be considered for the choice of the fluorescent labels.

 

For our complete range of fluorescent peptides, FRET-substrates and fluorescent building blocks visit shop.bachem.com

 

Peptides labeled with Tide Fluor and Tide Quencher as well as peptides and dye labeled peptides not contained in our catalogue can be requested from our Custom Synthesis Team.

 

 

References:

S. J. Bark and K. M. Hahn, Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: Peptides site-specifically labeled with two dyes, Methods 20 429-435 (2000)

D. Baechle et al., Biotinylated fluorescent peptide substrates for the sensitive and specific determination of cathepsin D activity, Journal of Peptide Science 11(3) 166-74 (2005)

D. Ozawa et al., Shuttling protein nucleolin is a microglia receptor for amyloid beta peptide 1-42, Biological and Pharmaceutical Bulletin 36(10) 1587–1593 (2013)

J. Domert et al., Spreading of amyloid-β peptides via neuritic cell-to-cell transfer is dependent on insufficient cellular clearance, Neurobiology of Disease 65 82–92 (2014)

K. Ikeda and K. Matsuzaki, Driving force of binding of amyloid beta-protein to lipid bilayers, Bichemical and Biophysical Research Communications 370(3) 525-9 (2008)

Y. Wang et al., Two-photon and time-resolved fluorescence conformational studies of aggregation in amyloid peptides, The Journal of Physical Chemistry B 114 7112–7120 (2010)

M. Citron et al., Generation of amyloid beta protein from its precursor is sequence specific, Neuron 14(3) 661-700 (1995)

D. M. Bickett et al., A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9), Analytical Biochemistry 212(1) 58-64 (1993)

E. Schmitt et al., Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells, Cell Death and Differentiation 5(6) 506-516 (1995)

FLUORESCENT PEPTIDES IN DEVELOPMENT FOR IMAGING

Fluorescently labeled peptides are useful for several applications including in vivo imaging, detecting enzyme activity and for visualizing cellular processes with fluorescence microscopy. In recent clinical and research studies, novel fluorescently labeled peptides are serving as innovative flashlights that light up cancer cells, nerves or other areas that require illumination. These labeled peptides may lead to a new generation of in vivo imaging tools for surgeons and provide for real-time imaging in surgery. Some examples of fluorescently labeled peptides for imaging that are in clinical development or have been published in research studies are shown in Table 1 below.

 

Product Name

Companies Involved

Highest Phase

Condition/Application

BLZ100

Blaze Bioscience Inc.

Morphotek

Fred Hutchinson Cancer Research Center

I

Brain Cancer, Skin Cancer, Solid Tumors

AVB620

Avelas Biosciences Inc.

I

Breast Cancer

KCC Peptide

University of Michigan, Olympus Corporation

I

Colon Polyps, Colorectal Cancer, Inflammatory Bowel Disease

EETI 2.5F

Stanford University

Research

Brain Cancer

F-NP41

University of California San Diego

Research

Surgery, Nerve Injury

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 FluorTM, 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.

MEET BACHEM: DR. MIRKO LOTZ, MARKETING SPECIALIST

PT: What is your official job title at Bachem?

Mirko: My official title is Marketing Specialist.

 

PT: How long have you been with Bachem?
Mirko: I joined the Global Marketing team at Bachem in March 2016. Before, I worked in Business Development for a company manufacturing industrial goods.

 

PT: Briefly, what do you do at Bachem?

Mirko: My work includes the update of technical brochures and information, literature research, maintenance and enrichment of product entries in our PIM system and the support in technical questions.

 

PT: What is your academic background?

Mirko: I studied biotechnology and afterwards I did my PhD in biophysics with the focus on integral membrane proteins.

 

PT: What do you like to do outside of work?

Mirko: I enjoy spending time with my family and meeting friends. I like bicycling and bicycles.

 

PT: What makes a perfect day for you?

Mirko: Having success and fun with the things I do.

 

PT: What do you like most about your job?

Mirko: It is a very multifaceted job, requiring a mixture of scientific working and “marketing thinking”. In addition, it is a very interdisciplinary work involving close communication with colleagues from diverging teams and departments, which I appreciate a lot.

 

PT: What do you do for fun?

Mirko: Being at the lake or seaside on a sunny day and enjoying a delicious food afterwards.

 

PT: What is your preferred peptide?

Mirko: Fluorescently labeled peptides, as they are the topic of some of my first projects.

 

 

PT: Thank you very much Mirko

Peptide Highlights

Interesting news about peptides in basic research and pharmaceutical development:

 

'Missing tooth' hydrogels handle hard-to-deliver drugs-ScienceDaily

Special, Life-Saving Delivery-Yale School of Medicine

Can Alzheimer's disease-associated peptide fight bacterial infection?-EurekAlert!

New discovery may improve future mosquito-Texas A&M University

Literature Citations

Bachem peptides and biochemicals are widely cited in research publications. Congratulations to all our customers with recent publications!

 

R. Richardson, M. Metzger, P. Knyphausen, T. Ramezani, K. Slanchev, C. Kraus, E. Schmelzer, and M. Hammerschmidt

Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals

Development 143, 2077 - 2088 (2016)

http://www.ncbi.nlm.nih.gov/pubmed/27122176

 

A. F Almeida-Santos, L. M. Kangussu, F..A. Moreira, R. A.S. Santos, D. C. Aguiar, and M. J. Campagnole-Santos

Anxiolytic- and antidepressant-like effects of angiotensin-(1–7) in hypertensive transgenic (mRen2)27 rats

Clin. Sci. 130, 1247 - 1255 (2016)

http://www.ncbi.nlm.nih.gov/pubmed/27129185

 

S. Delbary-Gossart, S. Lee, M. Baroni, I. Lamarche, M. Arnone, B. Canolle, A. Lin, J. Sacramento, E. A. Salegio, M.-N. Castel, N. Delesque-Touchard, A. Alam, P. Laboudie, B. Ferzaz, P. Savi, J.-M. Herbert, G. T. Manley, A. R. Ferguson, J. C. Bresnahan, F. Bono, and M. S. Beattie

A novel inhibitor of p75-neurotrophin receptor improves functional outcomes in two models of traumatic brain injury

Brain 139, 1762 - 1782 (2016)

http://brain.oxfordjournals.org/content/early/2016/04/15/brain.aww074

 

A. Z. Blatt, G. Saggu, K. V. Kulkarni, C. Cortes, J. M. Thurman, D. Ricklin, J. D. Lambris, J. G. Valenzuela, and V. P. Ferreira

Properdin-Mediated C5a Production Enhances Stable Binding of Platelets to Granulocytes in Human Whole Blood

J. Immunol. 196, 4671 - 4680 (2016)

http://www.ncbi.nlm.nih.gov/pubmed/27183616

 

B. Nuche-Berenguer, I. Ramos-Álvarez, and R. T. Jensen

Src kinases play a novel dual role in acute pancreatitis affecting severity but no role in stimulated enzyme secretion

Am. J. Physiol. Gastrointest. Liver Physiol. 310, G1015 - G1027 (2016)

http://www.ncbi.nlm.nih.gov/pubmed/27033118