Waldemar Waldeck

A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells.

To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.

Treatment of glioblastoma multiforme cells with temozolomide-BioShuttle ligated by the inverse Diels-Alder ligation chemistry

Recurrent glioblastoma multiforme (GBM), insensitive against most therapeutic interventions, has low response and survival rates. Temozolomide (TMZ) was approved for second-line therapy of recurrent anaplastic astrocytoma. However, TMZ therapy in GBM patients reveals properties such as reduced tolerability and inauspicious hemogram. The solution addressed here concerning GBM therapy consolidates and uses the potential of organic and peptide chemistry with molecular medicine. We enhanced the pharmacologic potency with simultaneous reduction of unwanted adverse reactions of the highly efficient chemotherapeutic TMZ. The TMZ connection to transporter molecules (TMZ-BioShuttle) was investigated, resulting in a much higher pharmacological effect in glioma cell lines and also with reduced dose rate. From this result we can conclude that a suitable chemistry could realize the ligation of pharmacologically active, but sensitive and highly unstable pharmaceutical ingredients without functional deprivation. The TMZ-BioShuttle dramatically enhanced the potential of TMZ for the treatment of brain tumors and is an attractive drug for combination chemotherapy.

NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

Replication of polyoma DNA in nuclear extracts and nucleoprotein complexes

Abstract Viral nucleoprotein complexes containing radioactive form l DNA or replicative intermediates were extracted from nuclei isolated from polyoma-infected 3T6 fibroblasts, pulse labelled with [3H]thymidine. Such extracts incorporated labelled dGTP into viral DNA, similar to intact isolated nuclei, but at a decreased rate and for shorter periods. The two kinds of nucleoprotein complexes containing form l DNA or replicative intermediates were separated and purified. Each complex retained some capacity to incorporate labelled dGTP and this reaction was stimulated by ATP. The new DNA consisted mainly of short strands hydrogen-bonded to the template. With replicative intermediate complexes incorporation occurred at random into different parts of the viral DNA, while form l complexes incorporated dGTP preferentially into a region around the origin of replication. A crude preparation of T-antigen stimulated the incorporation. The amount of synthesis was low and it was not possible to decide with certainty whether some of the incorporation observed with form 1 complexes represented initiation of new rounds of replication or whether it represented elongation of early replicative intermediates.

SPPS resins impact the PNA-syntheses' improvement.

The personalized medicine, also documented as “individualized medicine”, is an effective and therapeutic approach. It is designed to treat the disease of the individual patient whose precise differential gene expression profile is well known. The trend in the biomedical and biophysical research shows important consequences for the pharmaceutical drug and diagnostics research. It requires a high variability in the design and safety of target-specific pharmacologically active molecules and diagnostic components for imaging of metabolic processes. A key technology which may fulfill the highest demands during synthesis of these individual drugs and diagnostics is the solid phase synthesis which is congenial to automated manufacturing. Additionally the choice of tools like resins and reagents is pivotal to synthesize drugs and diagnostics in high quality and yields. Here we demonstrate the solid phase synthesis effects dependent on the choice of resin and of the deprotection agent.

Hybridization method for quantitation of replicating polyoma DNA sequences

Abstract Polyoma DNA was cleaved with restriction endonuclease HpaII, the fragments were separated by gel electrophoresis and transferred in good yield to separate nitrocellulose filters by a modification of the procedure of E. M. Southern (1975, J. Mol. Biol.98, 503–517). The filters were then used in hybridization experiments to localize the isotope in different parts of the polyoma genome after in vitro incorporation of labeled deoxyribonucleoside triphosphates into the DNA.