Grith Hagel

Solid-phase synthesis of smac peptidomimetics incorporating triazoloprolines and biarylalanines.

Apoptotic induction mechanisms are of crucial importance for the general homeostasis of multicellular organisms. In cancer the apoptotic pathways are downregulated, which, at least partly, is due to an abundance of inhibitors of apoptosis proteins (IAPs) that block the apoptotic cascade by deactivating proteolytic caspases. The Smac protein has an antagonistic effect on IAPs, thus providing structural clues for the synthesis of new pro-apoptotic compounds. Herein, we report a solid-phase approach for the synthesis of Smac-derived tetrapeptide libraries. On the basis of a common (N-Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide-alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities and yields (both typically above 90%). The peptides were subjected to biological evaluation in a live/dead cellular assay which revealed that structural decorations on the AVPF sequence indeed are highly important for cytotoxicity toward HeLa cells.

Ralph F. Hirschmann award address 2009: Merger of organic chemistry with peptide diversity

A huge unleashed potential lies hidden in the large and diverse pool of encoded and particularly nonencoded chiral α‐, β‐, and γ‐amino acids available today. Although these have been extensively exploited in peptide science, the community of organic chemistry has only used this source of diversity in a quite focused and targeted manner. The properties and behavior of peptides as functional molecules in biology are well documented and based on the ability of peptides to adapt a range of discrete conformers at a minimal entropic penalty and therefore ideally fitting their endogenous targets. The development of new organic reactions and chemistries that in a general and quantitative way transform peptides into new functional molecules, preferably on solid support, is a source of completely new classes of molecules with important and advantageous functional properties. The peptide diversity and the ability to perform chemistry on solid support add tremendously to the combinatorial scope of such reactions in pharmaceutical and materials screening scenario. In recent years, the need for “click” reactions to shape complex molecular architecture has been realized mainly with a basis in the world of peptides and DNA, and in polymer chemistry where connection of highly functionalized biologically active substances or property bearing fragments are assembled as molecular LEGO® using quantitative and orthogonal click chemistries. In this article, three such new reactions originating in the Carlsberg Laboratory over the last decade taking advantage of organic transformations in the peptide framework is presented. Initially, the click reaction between azide and terminal alkynes catalyzed by Cu(1) (CuAAC‐reaction) is described. This CuAAC “click” reaction was observed first at Carlsberg Laboratory in reactions of azido acid chlorides with alkynes on solid support. Second, the Electrophilic Aromatic Substitution Cyclization–Intramolecular Click‐Cascade (EASCy‐ICC) reaction will be presented. This quantitative stereo‐selective cascade reaction provides a highly diverse set of interesting novel scaffolds from peptides. Finally, we describe the preparation of solid phase peptide phosphine‐ and carbene‐based green catalysts (organozymes), which upon complex formation with transition metal perform with high turnovers under aqueous conditions. These catalysts thrive from the peptide folding and diversity, while phosphines and carbenes in the backbone provide for bidental complex formation with transition metals in a format providing an excellent entry into combinatorial catalyst chemistry.

Metabolically Stable Cellular Adhesion to Inert Surfaces

The structure of D‐amino acid hexapeptides that promote cellular adhesion was determined by screening D‐amino acid hexapeptide libraries synthesized on otherwise inert beaded PEGA resin. These new adhesion molecules provide a completely stable cellular environment and facilitate the maintenance of a monolayer of cells on beads for extended periods. The presence of the peptides promotes spreading of the cells on the bead surface. Not surprisingly, the molecules contained a significant number of arginines and/or lysines. However, the exact structure of each peptide is quite important for the degree of adhesion observed, and a motif with three or four basic amino acids spaced within amino acids of intermediate polarity clearly prevailed, for example, k‐l/r‐h‐r‐i/v‐r‐a; this maintains a polar/hydrophobic balance.

Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient’s tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine.

Abstract 2020: Spheroid culture of primary colorectal cancer cells from liver metastases as an in vitro model of patient tumors

More than 65 % of patients with colorectal cancer require chemotherapeutic treatment during the course of their disease. However, colorectal tumors demonstrate pronounced heterogeneity, leading to significant individual variation in sensitivity to standard chemotherapeutic drugs. Tools for assessing chemosensitivity and tailoring therapeutic regimens to the individual patient are therefore urgently needed. A promising approach is to isolate viable cancer cells from patient tissue and to test the chemosensitivity of the individual tumor in vitro. The aim of the current study was to establish primary colorectal cancer cell cultures from patient liver metastases and investigate if the cultured cells retained important characteristics of the original tumor. Small cell clusters were isolated from metastatic tissue and allowed to form cell spheroids in suspension culture. The cell composition of patient spheroids was characterized by immunostaining with cell type specific antibodies. Cancer stem cells were identified by staining for the markers CD44, CD166 and Lgr5. Spheroid size was monitored over time and growth curves established for each patient. The distribution of proliferating and apoptotic cells in the spheroids was assessed by staining for specific markers. Overall, the 3-D culture system was verified as a tumor model by comparing the results obtained for the spheroids to paraffin-embedded sections of the original tumors. Successful spheroid culture was obtained from more than 80 % of tissue samples. Immunostaining confirmed that the primary cultures were composed of epithelial cells with minimal contamination by other cell types. Cell morphology and expression of intestinal markers confirmed their malignant nature and colorectal origin. The dominating cell populations of the original tumor could generally be identified in the derived spheroids as well. The spheroids demonstrated significant growth in vitro with no decrease in proliferation or increase in apoptosis during short-term culture. In summary, spheroid cultures of colorectal cancer cells can be established from liver metastases of individual patients with a high success rate and the spheroids retain important cellular characteristics of the original tumor. Spheroid culture therefore represents a promising technique for establishing primary cultures for chemotherapeutic screening of the individual patient. Citation Format: Maria Jeppesen, Grith Hagel, Ben Vainer, Henrik Harling, Ole Thastrup, Lars Nannestad Jorgensen, Jacob Thastrup. Spheroid culture of primary colorectal cancer cells from liver metastases as an in vitro model of patient tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2020. doi:10.1158/1538-7445.AM2014-2020

Abstract 2718: Spheroid culture of colorectal cancer cells from patient tumor tissue preserves important cellular characteristics of the parental tumor.

At the time of diagnosis more than 65% of patients with colorectal cancer have advanced disease and require chemotherapeutic treatment. However, colorectal tumors demonstrate pronounced heterogeneity, leading to significant individual variation in sensitivity to standard chemotherapeutic drugs. Tools for assessing chemosensitivity and tailoring therapeutic regimens to the individual patient are therefore urgently needed. A promising approach is to isolate viable cancer cells from patient tissue and to test the chemosensitivity of the individual tumor in vitro. The aim of the current study was to establish primary colorectal cancer cell cultures from patient tissue and investigate if the cultured cells retain important characteristics of the original tumor. Small cell clusters were isolated from colorectal tumor tissue and allowed to form cell spheroids in suspension culture. The cell type composition of patient spheroids was characterized by immunostaining with cell type specific antibodies. Spheroid size was monitored over time and growth curves established for each patient. The distribution of proliferating and apoptotic cells in the spheroids was assessed by staining for specific markers. The 3D culture system was verified as a tumor model by comparing the results obtained for the spheroids to paraffin-embedded sections of the original tumors. Successful spheroid culture was obtained from more than 80% of tissue samples. Immunostaining confirmed that the primary cultures were composed of colorectal cancer cells, without significant contaminations by other cell types. Growth could be observed as an increase in spheroid size. The dominating cell populations of the original tumor could generally be identified in the derived spheroids as well. In summary, spheroid cultures of colorectal cancer cells can be established from tumor tissue of individual patients with a high success rate and the formed spheroids retain important cellular characteristics of the original tumor. Culture of colorectal cancer cells as spheroids represents a promising method for establishing primary cultures for chemotherapeutic screening of the individual patient. Citation Format: Maria Jeppesen, Grith Hagel, Anders Glenthoj, Ole Thastrup, Lars Nannestad Jorgensen, Jacob Thastrup. Spheroid culture of colorectal cancer cells from patient tumor tissue preserves important cellular characteristics of the parental tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2013-2718