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DYE LABELED PEPTIDES FOR CANCER RESEARCH
A major difference between dyes for in vitro and in vivo use are the wavelengths of emission and excitation. Typically, fluorescent dyes for in vitro applications excite and emit in the visible range, at wavelengths between 400 nm and 600 nm . However, light in the visible range does not have deep tissue penetration because of autofluorescence and the scattering nature of tissue as well as high levels of endogeneous absorbers such as hemoglobin, deoxyhemoglobin, water and lipids [2 – 4]. Therefore, fluorescent probes for in vivo studies commonly excite and emit in the near-infrared (NIR) range of light, at wavelengths between 700 nm and 1000 nm, at which these effects are less pronounced. These dye labeled molecules are also called near-infrared fluorescence (NIRF) imaging probes [2, 4].
Investigating Proteases Involved in Cancer
It is already well established that receptor-binding peptides can serve as carriers for labels and cytotoxic cargo for visualization or treatment of tumors, adopting Paul Ehrlich`s concept of the “magic bullet” [5, 6]. In contrast, protease inhibitors have failed thus far as cancer drugs, although our understanding of the tumor “degradome”, the repertoire of proteases and their natural inhibitors and interaction partners, advanced. Knowing which proteases are active in the tumor micro-environment may render it possible to tackle cancers with the use of protease-activated prodrugs (PAPs) . Dye labeled peptides are used as substrates for assaying the activity of proteases associated with the development and progression of cancer, and some examples are discussed in the following.
Matrix metalloproteinases (MMPs) are a family of related calcium-dependent zinc-containing endopeptidases, subdivided on the basis of substrate selectivity, domain assembly and conservation into collagenases, gelatinases, stromelysins, matrilysins, membrane-type and other MMPs . The family has at least 28 members in vertebrates, with 24 members in mammals and 23 members in human. Although they are indispensable for numerous physiological processes, they also carry out angiogenesis, promote tumor development and thus contribute to the spreading of cancer cells within the body .
For measuring the activity of MMPs, a convenient and sensitive approach is Fluorescence (or Förster) Resonance Energy Transfer- or “FRET”-measurement. FRET-substrates typically contain a fluorophore (donor), which is excited with light from an adequate source. The donor fluorescence is absorbed and thus attenuated (quenched) by a second, non-emitting chromophore (quencher), separated from the donor by the cleavage site. Upon proteolytic cleavage, the quenching is relieved and a signal can be measured as fluorescence increase [9 – 11]. As variations on this, the fluorescence of amino acids like tryptophan, contained in the peptide substrate sequence itself can serve as donor, described for example in a report about the optimization of collagenase substrates , or, the second chromophore can be fluorescent. Today, a broad range of fluorescent peptide substrates is available as catalog products, with diverging kinetic values for different MMPs.
Cysteine-dependent aspartate specific proteases (caspases) are key players in apoptosis, and their activation is generally considered as the “point of no return” in apoptotic pathways. Excessive apoptosis can lead to neurodegenerative diseases and ischemic injuries, whereas cancer and autoimmune diseases can result from insufficient apoptosis [13, 14]. Fluorescent labeled caspase substrates typically contain four amino acid residues with a stringent preference for an aspartic residue left (towards the N-terminus) and a methylamine right (towards the C-terminus) from the cleavage site, as described for instance for the caspase interleukin-Iβ-converting enzyme (ICE) . However, many variations of this sequence exist in order to increase the specificity for particular caspases, and only a small selection of literature examples can be given here [16 – 18].
Cathepsins are a family of lysosomal proteases, including cysteine, serine and aspartic proteases. They cleave their substrates in a relatively unspecific manner and are involved in numerous physiological and pathological processes, the latter including cancer . Cathepsins play a role in cell invasion and metastasis, and are considered as targets for cancer therapy . A dye labeled peptide was developed for example as a fluorogenic substrate for cathepsin D, which has been implicated in pathological processes such as Alzheimer`s Disease and breast cancer. The substrate is designed for FRET-experiments and resulted from a larger set of synthesized substrate sequences, which were evaluated against the background of enzymatic turnover kinetics .
Labeled RGD Peptides
The arginine-glycine-aspartic acid (RGD) sequence was discovered as a cell attachment site in fibronectin. Nowadays, RGD-peptides have become a popular tool for imaging αvβ3-integrin expressing tumor vasculature . Cyclization is commonly employed to improve the binding properties and stability of RGD peptides . The drug cilengitide, relying on a cyclic RGD peptide structure, was tested for treatment of glioblastoma, although finally did not alter patterns of progression in clinical trials .
As a recent development, proprietary Tide Fluor™ dyes are incorporated into cyclic RGD sequences. This group of dyes shows efficient excitation with light from common light sources and exhibits stronger fluorescence and significantly higher photo-stability, than most conventional or proprietary dyes. For example, a Tide Fluor™ labeled, cyclic RGD peptide is available, absorbing and emitting light in the NIR range.
There is an urgent demand for efficient and specific probes for imaging and assaying of molecules in cancer research. Fluorescence technology is applied in a broad range of fields, including culture assays, cell imaging, tissue staining, and in vivo studies. Dye labeled peptides as fluorescent substrates are particular valuable tools for the investigation of protease activity, leading to the formation of tumors or progression of cancer, and hence may represent a key technology for the development of cancer drugs. Numerous fluorescent peptide substrates are described in the literature and available as ready-made products. As a newer development, cyclic RGD peptides labeled with particularly strong emitting and stable dyes may contribute significantly to our understanding of cancer.
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(1) World cancer report, B.W. Stewart and C.P. Wild, Editors. 2014, World Health Organization – International Agency for Research on Cancer: 69372 Lyon Cedex 08, France. p. 495-502.
(2) C.H. Tung, Fluorescent peptide probes for in vivo diagnostic imaging. Biopolymers, 76(5), 391-403 (2004)
(3) S. Achilefu, Lighting up tumors with receptor-specific optical molecular probes. Technol. Cancer Res. Treat., 3(4), 393-409 (2004)
(4) J. Rao et al., Fluorescence imaging in vivo: recent advances. Curr. Opin. Biotechnol., 18(1), 17-25 (2007)
(5) P. Ehrlich, The collected papers of Paul Ehrlich, F. Himmelweite, M. Marquardt, and H. Dale, Editors. 1956, Pergamon, Elmsford, New York. p. 596-618.
(6) M. Langer and A.G. Beck-Sickinger, Peptides as carrier for tumor diagnosis and treatment. Curr. Med. Chem. Anticancer Agents, 1(1), 71-93 (2001)
(7) J. Vandooren et al., Proteases in cancer drug delivery. Adv. Drug Deliv. Rev., 97144-55 (2016)
(8) A. Khalid and M.A. Javaid, Matrix metalloproteinases: new targets in cancer therapy. J. Cancer Sci. Ther., 8(6), 143-153 (2016)
(9) D.M. Bickett et al., A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). Anal. Biochem., 212(1), 58-64 (1993)
(10) B. Beekman et al., Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay. FEBS Lett., 418(3), 305-9 (1997)
(11) M.O. Palmier and S.R. Van Doren, Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect. Anal. Biochem., 371(1), 43-51 (2007)
(12) J. Berman et al., Rapid optimization of enzyme substrates using defined substrate mixtures. J. Biol. Chem., 267(3), 1434-7 (1992)
(13) S. Shalini et al., Old, new and emerging functions of caspases. Cell. Death Differ., 22(4), 526-39 (2015)
(14) S.M. Man and T.D. Kanneganti, Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat. Rev. Immunol., 16(1), 7-21 (2016)
(15) N.A. Thornberry et al., A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature, 356(6372), 768-74 (1992)
(16) D.W. Nicholson et al., Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature, 376(6535), 37-43 (1995)
(17) J. Xiang et al., BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc. Natl. Acad. Sci. USA, 93(25), 14559-63 (1996)
(18) P.S. Schwartz and D.J. Waxman, Cyclophosphamide induces caspase 9-dependent apoptosis in 9L tumor cells. Mol. Pharmacol., 60(6), 1268-79 (2001)
(19) S. Conus and H.U. Simon, Cathepsins and their involvement in immune responses. Swiss Med. Wkly., 140w13042 (2010)
(20) T. Nomura and N. Katunuma, Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells. J. Med. Invest., 52(1-2), 1-9 (2005)
(21) S.V. Gulnik et al., Design of sensitive fluorogenic substrates for human cathepsin D. FEBS Lett., 413(2), 379-84 (1997)
(22) K. Temming et al., RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature. Drug Resist. Updat., 8(6), 381-402 (2005)
(23) G. Eisele et al., Cilengitide treatment of newly diagnosed glioblastoma patients does not alter patterns of progression. J. Neurooncol., 117(1), 141-5 (2014)
Fluorescent dye labeled peptides may lead to a new generation of diagnostic and imaging tools for use in oncology applications. Novel dye labeled peptides are being developed as innovative flashlights to guide surgeons during cancer surgery and as tools to improve cancer screening and diagnosis. In the area of oncology, several dye labeled peptides are already in clinical development for use in diagnostic and imaging applications as shown in Table 1 below.
Table 1: Selection of Dye Labeled Peptides in Development for Diagnostics and Imaging
|Product Name||Companies Involved||Highest Phase||Condition/Application|
|AVB620||Avelas Biosciences Inc.||II||Breast Cancer|
|ABY-029||Dartmouth-Hitchcock Medical Center, Affibody AB, LI-COR Biosciences, University of Alabama at Birmingham (UAB) Vector Production Facility||I||Glioma|
|BLZ100||Blaze Bioscience Inc.||I||Cancer Surgery|
|KCC Peptide||University of Michigan, Olympus Corporation||I||Colon Polyps, Colorectal Cancer, Inflammatory Bowel Disease|
|EMI-137||Edinburgh Molecular Imaging, University Medical Center Groningen||I||Barrett Esophagus, Esophageal Cancer, Dysplasia in Barrett Esophagus|
|KSP-910638G Heptapeptide||University of Michigan, Edinburgh Molecular Imaging Ltd||I||GI Malignancies|
|QRH-882260 Heptapeptide||University of Michigan||I||Colon Cancer Prevention|
Avelas Biosciences is developing AVB620 as an in vivo surgical diagnostic agent for breast cancer. AVB620 is a fluorescent protease-activated peptide that detects, marks and diagnoses cancer. AVB620 is administered before surgery and imaging is performed with a fluorescence imaging camera. The technology is designed to enable surgeons to identify critical cancer margins and stage lymph nodes (1). In May 2016, Avelas reported interim results from the Phase Ib trial of AVB620. No drug-related adverse events were noted in the trial and AVB620 was able to achieve separation of tumor from adjacent tissues using a fluorescent image in both primary tumors and in lymph nodes. In July 2017, Avelas initiated a Phase II study of AVB620 in women with breast cancer undergoing surgery (2).
Dartmouth has partnered with Affibody AB, LI-COR Biosciences and the University of Alabama at Birmingham (UAB) Vector Production Facility to develop and produce ABY-029 as a probe to guide surgery. ABY-029 binds to cancer cell receptors and highlights tumors (3). The product is an IRDye® 800CW Maleimide labeled Affibody peptide. Dartmouth is currently recruiting participants for a Phase I trial in patients with recurrent glioma. The primary objective of the study is to determine if micro-dose injections of ABY-029 lead to detectable signals in sampled tissues (4).
BLZ100 (tozuleristide) is 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. BLZ100 is used to “paint” tumors in order to guide surgeons during cancer removal. 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 (2). 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 (5).
KCC Peptide contains 7 amino acids and is attached to FITC (Fluorescein isothiocyanate). Researchers at the University of Michigan are developing KCC Peptide for use during colonoscopy procedures. 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. In 2016, the University of Michigan completed a Phase Ib study with KCC Peptide to determine if the peptide glows sufficiently (6).
Edinburgh Molecular Imaging has developed a fluorescent tracer, EMI-137, that specifically targets the biomarker c-Met. The tracer is a small peptide labeled with a fluorophore. University Medical Center Groningen is currently studying EMI-137 in a Phase I trial that aims to detect pre-malignant lesions in patients with Barrett’s Esophagus (7).
The University of Michigan is currently recruiting participants for a Phase Ia study of KSP-910638G Heptapeptide to evaluate its safety for detection of neoplastic tissues in the gastrointestinal tract. KSP-910638G is a fluorescently labeled peptide that consists of a heptapeptide attached via a five amino acid linker to a near-infrared fluorophore, IRDye® 800CW. This peptide is specific for human epithelial growth factor receptor 2 (HER2) (8).
In addition, the University of Michigan is recruiting participants for a Phase Ib study of QRH-882260 Hepatapeptide, a peptide attached via a 5 amino acid linker to a near-infrared fluorophore Cy5. The study will test the safety and efficacy of QRH-882260 Heptapeptide when administrated to patients undergoing colonoscopy for endoscopic resection of colonic adenomas or for dysplasia surveillance in patients with irritable bowel disease (IBD) (9).
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 dye labeled peptides for research and clinical applications, Bachem provides a one-stop shop for the custom synthesis of labeled 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 FluorTM dyes are optimized and designed to meet the highest demands in fluorescent intensity and photo-stability and outperform most conventional and proprietary dyes. We also offer dye labeled catalog products in our online shop such as item H-8322 Cyclo(-Arg-Gly-Asp-D-Tyr-Lys(Tide Fluor™ 7WS)). For custom synthesis, please contact our Custom Synthesis team to request a quote for your labeled peptide or amino acid.
(1) AVB-620, Avelas Biosciences (2017)
(2) Medtrack (2017)
(3) Dartmouth wins FDA approval for aid to guide cancer surgery, Dartmouth News (2016)
(4) A microdose evaluation study of ABY-029 in recurrent glioma (ABY-029), ClinicalTrials.gov (2017)
(5) Clinical Trials, Blaze Bioscience (2017)
(6) Study of KCC Peptide application in the colon (KCC 1B), ClinicalTrials.gov (2016)
(7) Molecular fluorescence endoscopy of (pre)malignant esophageal lesions (EAGLE), ClinicalTrials.gov (2017)
(8) Study of the safety of KSP Heptapeptide (KSP-910638G), ClinicalTrials.gov (2017)
(9) Study of QRH-882260 Heptapeptide application in the colon, ClinicalTrials.gov (2017)
What is your official job title at Bachem?
How long have you been with Bachem? Where did you work before Bachem?
I started working at Bachem in September 2016. I started my professional path as a Customer Service Manager at Solvay Pharmaceuticals Marketing & Licensing AG responsible for customers within the area of Latin America. After an insight in a trust company and the start of my part-time studies, I decided to come back to the life sciences field and used to work as a procurement manager for Acino Pharma AG.
Briefly, what do you do at Bachem?
I am a Sales Representative for research-grade materials.
What is your academic background/degrees or training?
I studied Business Administration with a major in Management & Entrepreneurship at the School of Business of the University of Applied Sciences Northwestern Switzerland. I decided to study in a part-time model, which means that I was working and studying at the same time for 4 years.
What do you like to do outside of work (interests, hobbies)?
I like all different kind of sports but my big passion is boxing. Besides training for myself, I am also a voluntary boxing coach for children and teenagers in a small regional Boxing Gym. What I also enjoy a lot is to cook, to read and to go out with my close friends to enjoy time off.
What do you like most about your job?
No day is like the other in our job as Sales Representatives, but this is what makes it exciting. Even being with Bachem for a year now, there is always a challenging task and something new to learn which helps me to grow – not just professionally but also as a person – while interacting with all kind of people and experiencing new situations.
Do you like to communicate any key message to the reader?
Reflect on yourself. In my opinion, self-reflection has a huge positive impact on how we work together and treat each other, which is a precondition to set new standards and stay the pioneering partner for peptides.
Thank you very much Mesut.
Interesting news about peptides in basic research and pharmaceutical development:
New study discovers “killer peptide” that helps eliminate resistant cancer cells-University of Kentucky