Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

Background Natural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. Methods The study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids. Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. Results The results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. Conclusions We report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. General significance Understanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors

Studies on interactions of protooncogenic Gquadruplexes with ligands

Wydział ChemiiG-kwadrupleksy są formą DNA, tworzącą się poprzez zwinięcie się jednej lub wielu nici DNA bogatych w reszty guanozynowe. Struktury te zbudowane są z kwartetów guaninowych. Gkwadrupleksy odgrywają istotną role w w ciele człowieka, a ponadto uważa się iż ich stabilizacja może odegrać kluczową rolę w nowych terapiach przeciwnowotworowych. Protoonkogeny ludzkie są bogate w zasady guaninowe, dzięki czemu możliwe jest na tych odcinkach tworzenie struktur G-kwadrupleksowych. Sugeruje się, że stabilizacja tych struktur poprzez oddziaływanie z ligandami może prowadzić do inhibicji procesów kancerogennych, jak i nadekspresji genów na tych odcinkach. Celem rozprawy doktorskiej było zbadanie oddziaływań pomiędzy G-kwadrupleksami tworzonymi na odcinkach protoonkogenowych i ligandami karbazolowymi. W tym celu przeprowadzono szereg obliczeń z zastosowaniem mechaniki kwantowej oraz modelownia molekularnego. Symulacje dynamiki molekularnej zostały przeprowadzone dla lepszego wyjaśnienia przebiegu oddziaływań badanych ligandów z receptorem. Obliczenia kwantowomechaniczne pozwoliły określić swobodę konformacyjną ligandów. Uzyskane rezultaty wskazują, iż badane ligandy karbazolowe stabilizują G-kwadrupleksy tworzące się na odcinkach protoonkogenowych, czyniąc je atrakcyjnymi strukturami do dalszych prac w celu poprawy ich właściwości.Guanines can self-associate in a square co-planar array to form G-quartets. G-quartets can self-stack resulting in the formation of four-stranded DNA. Gquadruplex structures play important roles in human body and are viewed as valid therapeutic targets in human cancer disease. Human protooncogenes c-MYC, C-KIT and BCL-2 are rich in guanine, which enable the formation of Gquadruplexes. Stabilization of these structures can inhibit tumor growth by inhibition of the overexpression of genes were they can be formed; proto-oncogenes are considering as attractive targets in developing new therapies. The aim of this doctoral dissertation was to study the interactions between G-quadruplexes formed by oligonucleotides corresponding to promoter regions of oncogenes and three carbazole ligands. In the course of these studies quantum mechanical and molecular calculations were performed to investigate the interactions. Molecular dynamics simulations were carried out to determine the atomistic detail of ligand binding. Quantummechanical calculations allowed the determination of conformational flexibility of the ligand. The results indicate that studied carbazole ligands are able to stabilize the G-quadruplexes and also that carbazole skeleton is an attractive scaffold for further structural modifications

Studies on interactions of protooncogenic Gquadruplexes with ligands

Wydział ChemiiG-kwadrupleksy są formą DNA, tworzącą się poprzez zwinięcie się jednej lub wielu nici DNA bogatych w reszty guanozynowe. Struktury te zbudowane są z kwartetów guaninowych. Gkwadrupleksy odgrywają istotną role w w ciele człowieka, a ponadto uważa się iż ich stabilizacja może odegrać kluczową rolę w nowych terapiach przeciwnowotworowych. Protoonkogeny ludzkie są bogate w zasady guaninowe, dzięki czemu możliwe jest na tych odcinkach tworzenie struktur G-kwadrupleksowych. Sugeruje się, że stabilizacja tych struktur poprzez oddziaływanie z ligandami może prowadzić do inhibicji procesów kancerogennych, jak i nadekspresji genów na tych odcinkach. Celem rozprawy doktorskiej było zbadanie oddziaływań pomiędzy G-kwadrupleksami tworzonymi na odcinkach protoonkogenowych i ligandami karbazolowymi. W tym celu przeprowadzono szereg obliczeń z zastosowaniem mechaniki kwantowej oraz modelownia molekularnego. Symulacje dynamiki molekularnej zostały przeprowadzone dla lepszego wyjaśnienia przebiegu oddziaływań badanych ligandów z receptorem. Obliczenia kwantowomechaniczne pozwoliły określić swobodę konformacyjną ligandów. Uzyskane rezultaty wskazują, iż badane ligandy karbazolowe stabilizują G-kwadrupleksy tworzące się na odcinkach protoonkogenowych, czyniąc je atrakcyjnymi strukturami do dalszych prac w celu poprawy ich właściwości.Guanines can self-associate in a square co-planar array to form G-quartets. G-quartets can self-stack resulting in the formation of four-stranded DNA. Gquadruplex structures play important roles in human body and are viewed as valid therapeutic targets in human cancer disease. Human protooncogenes c-MYC, C-KIT and BCL-2 are rich in guanine, which enable the formation of Gquadruplexes. Stabilization of these structures can inhibit tumor growth by inhibition of the overexpression of genes were they can be formed; proto-oncogenes are considering as attractive targets in developing new therapies. The aim of this doctoral dissertation was to study the interactions between G-quadruplexes formed by oligonucleotides corresponding to promoter regions of oncogenes and three carbazole ligands. In the course of these studies quantum mechanical and molecular calculations were performed to investigate the interactions. Molecular dynamics simulations were carried out to determine the atomistic detail of ligand binding. Quantummechanical calculations allowed the determination of conformational flexibility of the ligand. The results indicate that studied carbazole ligands are able to stabilize the G-quadruplexes and also that carbazole skeleton is an attractive scaffold for further structural modifications

Carbazole Derivatives’ Binding to c-KIT G-Quadruplex DNA

The binding affinities of three carbazole derivatives to the intramolecular G-quadruplex (GQ) DNA formed by the sequence 5′-AGGGAGGGCGCTGGGAGGAGGG-3′, derived from the c-KIT 1 oncogene region, were investigated. All carbazole cationic ligands that differed in the substituents on the nitrogen atom were able to stabilize G-quadruplex, as demonstrated using UV-Vis, fluorescence and CD spectroscopic techniques as well as molecular modeling. The spectrophotometric titration results showed spectral features characteristic of these ligands-bathochromic shifts and initial hypochromicity followed by hyperchromicity at higher GQ concentrations. All free carbazole ligands exhibited modest fluorescent properties, but after binding to the DNA the fluorescence intensity increased significantly. The binding affinities of carbazole ligands to the c-KIT 1 DNA were comparable showing values in the order of 105 M−1. Molecular modeling highlights the differences in interactions between each particular ligand and studied G-quadruplex, which potentially influenced binding strength. Obtained results relevant that all three investigated ligands have stabilization properties on studied G-quadruplex

Regioselective glycosylation of novobiocin alters activity

Glycosylation is a promising approach to overcome antimicrobial drug resistance. In this study, we investigated Koenigs-Knorr and phase transfer glycosylation on novobiocin. While the former only gave a 4′-OH product, the later produced mainly a kinetic controlled 5-OH product, but still achieved the 4′-OH modification and novoise-glycosylated products (with stronger base), as well as a diglycosylated compound. Investigation on the antibacterial activity indicate that the presence of galactose moiety helps to improve activity possibly via enhanced cellular uptake