Konjugate des Antisense-Oligonucleotids Oblimersen mit Estrogenrezeptor-α-Agonisten und - Antagonisten zur Anwendung gegen hormonresponsiven Brustkrebs

Moderne therapeutische Ansätze zur Behandlung seltener oder schwer zu kurierender Erkrankungen beinhalten immer mehr zielgerichtete Therapien. Dies kann durch Verwendung von organ-, gewebe- oder zellspezifischen Kleinmolekülen (engl. small molecules, SM) realisiert werden, welche definierte Rezeptoren auf Zellen zum Ziel haben. Die entsprechenden, kovalent an therapeutische Biooligomere wie z.B. DNA angebundenen Konjugate können über das Konzept des aktiven Targetings gezielt an den Ursprung von Krankheiten dirigiert wer-den. In der vorliegenden Arbeit wurden modifizierte Derivate des endogenen Estrogenrezeptor-Alpha-(ERα)-Agonisten Estradiol (E2) und Estron (E1) sowie solche, die von dem selektiven Estrogenrezeptor-Modulator (SERM) 4-Hydroxytamoxifen (4-OHT) abgeleitet sind, verwendet. Mit diesen Liganden wurde versucht, hormon-responsive Brustkrebszellen anzugreifen. Durch Verwendung hetero-bifunktionaler Spacer wurden die Liganden mit dem gegen das B-Zellen-Lymphom 2-Protein (BCl2) gerichtete therapeutische Antisense-Oligonucleotid (ASO) Oblimersen kovalent verbunden. Mit der kupferfreien, ringspannungs-vermittelten Azid-Alkin-Cycloaddition (SPAAC) an CPG-gebundenen, einzelsträngigen Oligonucleotiden (ssONs) konnte diese Konjugate postsynthetisch realisiert werden. Die Liganden wurden darüber hinaus mit einem fluoreszenzbasierten Verdrängungs-Assay auf ihre Rezeptoraffinität getestet. Final wurden die erhaltenen Konjugate auf ihre anti-proliferativen Eigen-schaften und ihre Fähigkeit, die Apoptose zu stimulieren, in Zellkultur untersucht. Zu diesem Zweck wurden die Verbindungen in ERα-überexprimierenden T47D-Zellen und ERα-defizitären HepG2-Zellen getestet und die biologische Wirksamkeit anhand der Caspase-Aktivität gemessen. Hierbei konnte Zellspezifität für ERα-überexprimierende T47D-Zellen nachgewiesen werden. Erste Untersuchungen zur Zellaufnahme der Konjugate wurden mittels Fluoreszenzmikroskopie in beiden Zelllinien durchgeführt. Die Konjugate zeigten eine mehrfache Steigerung der biologischen Aktivität im Vergleich zu etablierten Standards und zusätzlich eine erhöhte zelluläre Internalisierung im Vergleich zu nicht-konjugierten, einzelsträngigen Oligonucleotiden (ssONs), was mutmaßlich auf rezeptorvermittelte Endozytose zurückzuführen ist.Modern therapeutic approaches are developing towards targeted therapies of diseases. This can be achieved using organ/tissue/cell-specific small molecules (SM) for targeting certain cellular receptors in order to deliver covalently attached therapeutics specifically to the origin of diseases via the concept of active targeting. Herein, conjugates of the endogenous estrogen receptor alpha (ERα) agonists estradiol (E2) and estrone (E1) as well as such derived from the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) were chosen to target the estrogen receptor in hormone-responsive breast cancer cells. These ligands were covalently coupled to the therapeutic antisense oligonucleotide (ASO) oblimersen directed against BCl2 using strain-promoted azide-alkyne cycloaddition (SPAAC) post-synthetically on CPG-bound single-stranded oligonucleotides (ssONs). Ligands were subsequently tested for receptor affinity using a fluorescence-based competition assay. Additionally, the obtained conjugates were evaluated for their anti-proliferative properties and their ability to stimulate programmed cell death, i.e. apoptosis. For this purpose, the compounds were tested in ERα-overexpressing T47D cells as well as ERα-deficient HepG2 cells and the biological efficacy was measured based on caspase activity. Initial studies on cellular uptake of the conjugates were performed using fluorescence microscopy in both cell lines. The conjugates showed a multiple in-crease in biological activity compared to established standards and additionally increased cellular internalization relative to non-conjugated ONs, which was presumably based on by receptor-mediated uptake. The synthesis of the key building blocks for ligand construction, solid-phase oligonucleotide synthesis (SPOS), ligand attachment to ONs and biological evaluation of resultant conjugates are presented in this dissertation.Modern therapeutic approaches are developing towards targeted therapies of diseases. This can be achieved using organ/tissue/cell-specific small molecules (SM) for targeting certain cellular receptors in order to deliver covalently attached therapeutics specifically to the origin of diseases via the concept of active targeting. Herein, conjugates of the endogenous estrogen receptor alpha (ERα) agonists estradiol (E2) and estrone (E1) as well as such derived from the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) were chosen to target the estrogen receptor in hormone-responsive breast cancer cells. These ligands were covalently coupled to the therapeutic antisense oligonucleotide (ASO) oblimersen directed against BCl2 using strain-promoted azide-alkyne cycloaddition (SPAAC) post-synthetically on CPG-bound single-stranded oligonucleotides (ssONs). Ligands were subsequently tested for receptor affinity using a fluorescence-based competition assay. Additionally, the obtained conjugates were evaluated for their anti-proliferative properties and their ability to stimulate programmed cell death, i.e. apoptosis. For this purpose, the compounds were tested in ERα-overexpressing T47D cells as well as ERα-deficient HepG2 cells and the biological efficacy was measured based on caspase activity. Initial studies on cellular uptake of the conjugates were performed using fluorescence microscopy in both cell lines. The conjugates showed a multiple in-crease in biological activity compared to established standards and additionally increased cellular internalization relative to non-conjugated ONs, which was presumably based on by receptor-mediated uptake. The synthesis of the key building blocks for ligand construction, solid-phase oligonucleotide synthesis (SPOS), ligand attachment to ONs and biological evaluation of resultant conjugates are presented in this dissertation