AN INVESTIGATION OF THE ELECTRONIC-STRUCTURE OF THE ANTITUMOR DRUG ELLIPTICINE AND ITS DERIVATIVES .1. GEOMETRICAL AM1 STUDY

Ellipticine and its derivatives are a class of molecules that show antitumor and cytotoxic activity with a multimodal mechanism of action. In this paper we report a preliminary Austin Method One (AM1) study of ellipticine and some molecules derived from it. We have observed a relationship between charge density distribution and biological selectivity. A mechanism that could improve cytotoxic activity is proposed.

Why further investigation of brazilian Strychnos?

During the past fifteen years, African Strychnos species have been pharmacologically and chemically screened by some European Universities, sometimes with the collaboration of some African Institutes. Strychnos usambarensis will be discussed here because of our interest in it, after the discovery of its use in the preparation arrow poison with curarizing activity.The European studies have show thah the African species of Strychnoschemically are very similar to certain South-American species. Indeed, the occurrence in the African species of curarizing quaternary bases, and in the American species of tetanizing tertiary bases, such as akagerine, emphasizes the unity of genus Strychnos in terms of it alkaloids. Many biological activities (antmicrobial, hypotensive, smasmolytic, amoebicial...) have been detected.Finally, the antimitotic properties of some alkaloids will be presented. Besides know antitumor drugs, e.g. alstonine and ellipticine, African species possess new antimitotic products, e.g. strychnopentamine present in Strychnos usambarensis.Because of these findings, the main reasons why further investigate Brazilian Strychnos species are the lack of knowledge about:- the distribution on the species in the southern part of the great Amazon Basin.Accordin to Krukoff, it is possible that new species may be found there.- the distribution of alkaloids in many of know species. Indeed, there is little information on the variation in compposition of the alkaloidal mixtures in the different parts of the plants.- the biological of alkaloids. In the past, interest has been focused too narrowly on possible curarizing properties.Why should Brazilian species not cantain alkaloids with properties similar to those found in the African species?

In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants

In the present study, a quassinoid, neosergeolide, isolated from the roots and stems of Picrolemma sprucei (Simaroubaceae), the indole alkaloids ellipticine and aspidocarpine, isolated from the bark of Aspidosperma vargasii and A. desmanthum (Apocynaceae), respectively, and 4-nerolidylcatechol, isolated from the roots of Pothomorphe peltata (Piperaceae), all presented significant in vitro inhibition (more active than quinine and chloroquine) of the multi-drug resistant K1 strain of Plasmodium falciparum. Neosergeolide presented activity in the nanomolar range. This is the first report on the antimalarial activity of these known, natural compounds. This is also the first report on the isolation of aspidocarpine from A. desmanthum. These compounds are good candidates for pre-clinical tests as novel lead structures with the aim of finding new antimalarial prototypes and lend support to the traditional use of the plants from which these compounds are derived.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

Estrutura e propriedades de elipticinas

The ellipticines constitute a broad class of molecules with antitumor activity. In the present work we analyzed the structure and properties of a series of ellipticine derivatives in the gas phase and in solution using quantum mechanical and Monte Carlo methods. The results showed a good correlation between the solvation energies in water obtained with the continuum model and the Monte Carlo simulation. Molecular descriptors were considered in the development of QSAR models using the DNA association constant (log Kapp) as biological data. The results showed that the DNA binding is dominated by electronic parameters, with small contributions from the molecular volume and area.

Alcaloides indólicos de cascas de Aspidosperma vargasii e A. desmanthum

Fractionation of extracts resulted in isolation and identification of indole alkaloids ellipticine and N-methyltetrahydroellipticine from A. vargasii and aspidocarpine from A. desmanthum. Identification of these compounds was achieved based on IR, MS, ¹H, 13C and 2-D NMR data and comparison to data in the literature.

Untersuchung der DNA-bindenden Eigenschaften neuer Nukleobasen-gekoppelter Pyrrolcarboxamide, Combilexine und Hetaren[a]carbazol-Derivate: Validierung und Etablierung von in-vitro- und in-silico-Methoden

Da maligne Neoplasien durch Mutationen in Proto-Onko- und/oder Tumorsuppressorgenen ausgelöst werden, stellt die DNA eines der wichtigsten Targets für die Entwicklung neuer Zytostatika dar. Auch bei den im Arbeitskreis Pindur designten und synthetisier-ten Verbindungen der Nukleobasen-gekoppelten Pyrrolcarboxamid-, der Hetaren[a]carbazol- und der Combilexin-Reihe handelt es sich um DNA-Liganden mit potentiell antitumoraktiven Eigenschaf-ten. Die einen dualen Bindemodus aufweisenden Combilexine bestehen aus einem Interkalator (u. a. Naphthalimid, Acridon), der über einen Linker variabler Kettenlänge mit einer rinnenbin-denden, von Netropsin abgeleiteten Bispyrrol-, oder einer bioisosteren Imidazol-, Thiazol- oder Thiophen-pyrrolcarboxamid-struktur verknüpft ist. Das N-terminale Ende der Combilexine wird von einer N,N-Dimethylaminopropyl- oder -ethyl-Seitenkette gebildet. Die DNA-Affinitäten der Liganden wurden mittels Tm-Wert-Messung-en bestimmt. Diese Denaturierungsexperimente wurden sowohl mit poly(dAdT)2- als auch mit Thymus-DNA (~42% GC-Anteil) durchge-führt, um Aussagen zur Stärke und zur Sequenzselektivität der DNA-Bindung machen zu können. Des Weiteren wurden die Bindekon-stanten einiger ausgewählter Vertreter mit Hilfe des Ethidium-bromid-Verdrängungsassays ermittelt; einige Testverbindungen wurden zudem auf potentiell vorhandene, TOPO I-inhibierende Eigenschaften untersucht. Diese biochemischen und biophysika-lischen Tests wurden durch Molecular Modelling-Studien ergänzt, die die Berechnung von molekularen Eigenschaften, die Durch-führung von Konformerenanalysen und die Simulation von DNA-Ligand-Komplexen (Docking) umfassten. Durch Korrelation der in vitro-Befunde mit den in silico-Daten gelang es, vor allem für die Substanzklasse der Combilexine einige richtungweisende Struktur-Wirkungsbeziehungen aufzustellen. So konnte gezeigt werden, dass die Einführung eines Imidazol-Rings in die rinnen-bindende Hetaren-pyrrolcarboxamid-Struktur der Combilexine aufgrund der H-Brücken-Akzeptor-Funktion des sp2-hybridisierten N-Atoms eine Verschiebung der Sequenzselektivität der DNA-Bindung von AT- zu GC-reichen Arealen der DNA bedingt. Zudem erwies sich ein C3-Linker für die Verknüpfung des Naphthalimids mit dem rinnenbindenden Strukturelement als am besten geeignet, während bei den Acridon-Derivaten die Verbindungen mit einem N-terminalen Buttersäure-Linker die höchste DNA-Affinität aufwiesen. Dies ist sehr wahrscheinlich auf die im Vergleich zum Naphthalimid-Molekül geringere y-Achsen-Ausdehnung (bzgl. eines x/y-Koordinatensystems) des Acridons zurückzuführen. Die ermittelten Struktur-Wirkungsbeziehungen können dazu herangezogen werden, das rationale Design neuer DNA-Liganden mit potentiell stärkerer DNA-Bindung zu optimieren.

A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like Replication Structures

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5′-tailed fork was more active than the 3′-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3′ to 5′ along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mm KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 μm and 2.2 μm, respectively) and ATPase (Ki approximately 1.3 μm and 3.0 μm, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

New indole alkaloids from the roots of Ochrosia acuminata

Two new indole alkaloids, polyneuridine-N-oxide (1) and 17-hydroxy-10-methoxy-yohimbane (2), together with seven known alkaloids were isolated from the roots of Ochrosia acuminata collected in Savu, Indonesia. 9-Methoxyellipticine (3) and ellipticine (4) were responsible for the antitumor activities of the extract. The structures of all compounds were elucidated using MS and NMR methods.

Isoellipticines: novel compounds for leukaemia treatment

Ellipticine, an anticancer agent, has had limited clinical success due to low solubility and toxic side effects. To overcome these limitations, a panel of novel ellipticine isomers were designed and synthesised with the aim of evaluating their anti-cancer effects on selected cancer cell lines. A preliminary NCI 60-cell screen demonstrated that these isoellipticines displayed promising anti-tumour activity across a number of different cell types, particularly leukaemia cell lines. We consequently examined the effect of these derivatives in detail on the Acute Myeloid Leukaemia (AML) cell line, MV4-11. Cell cycle analyses revealed that the compounds had a range of distinctive cell cycle effects on MV4-11 cells. 7-Hydroxyisoellipticine showed the most promise with respect to cytostatic activity. We demonstrated that this compound inhibited proliferation of leukaemia cells by preventing cells from progressing from G2 phase. Our research suggests that this is mediated by an induction of reactive oxygen species (ROS), which in turn activates the DNA damage response pathway. More extensive research on the source of ROS generated by the most potent derivative, 7-formyl-10-methylisoellipticine showed that this compounds cytotoxicity is partially mediated by an induction of mitochondrial derived reactive oxygen species (ROS). We showed that 7-formyl-10-methylisoellipticine has synergistic effects when used in combination with the clinically used AML drug, daunorubicin, as well as DPI, a Nox inhibitor. Additionally, combination experiments with other drugs served to give us a deeper insight into 7- formyl-10-methylisoellipticine mechanism of action. 7-Formyl-10-methylisoellipticine also displayed promising in vivo results. Treatment resulted in a lack of toxicity, as measured by body weight changes and liver enzyme analyses. Most importantly, 7-formyl-10-methylisoellipticine demonstrated potent anti-tumour activity in the in vivo xenograft mouse model, implying the potential of isoellipticines as novel chemotherapeutic agents in the treatment of leukaemia. In summary, this study provides for the first time detailed cellular information on the potential use of isoellipticines as chemotherapeutic agents. Our study documents for the first time, the therapeutic potential of an isoellipticine compound in a subcutaneous AML cell-derived xenograft (CDX) model. By probing the mechanism of action of this novel compound class we have uncovered a potential clinical application in the field of adjuvant therapy. We anticipate that the recent research on ellipticine derivatives, such as this study, will lead the development of an ellipticine analogue that may be employed clinically.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents

Ellipticine is a natural product which possesses multimodal anti-cancer activity. This thesis encompasses the synthesis and biological evaluation of novel ellipticine and isoellipticine derivatives as anti-cancer agents. Expanding on previous work within the group utilising vinylmagnesium bromide, derivatisation of the C5 position of ellipticine was accomplished by reaction of a key ketolactam intermediate with Grignard reagents. Corresponding attempts to introduce diverse substitution at the C11 position were unsuccessful, although one novel C11 derivative was produced using an alkyllithium reagent. A panel of novel ellipticinium salts encompassing a range of substitutions at the N2, C9 and N6 positions were prepared. Extensive derivatisation of the N10 position of isoellipticine was undertaken for the first time. Novel substitution in the form of acid and methyl ester functionalities were introduced at the C7 position of isoellipticine while novel C7 aldehyde and alcohol derivatives were synthesised. A large panel of isoellipticinium salts were prepared with conditions adjusted for the reactivity of the alkyl halide. Novel coupling reactions to increase the yield of isoellipticine were attempted but proved unsuccessful. A panel of 54 novel derivatives was prepared and a multimodal analysis of their anti-cancer activity was conducted. The NCI 60-human tumour cell lines screen was a primary source of information on the in vitro activity of compounds with derivatives found to exert potent anticancer effects, with mean GI50 values as low as 1.01 μM across the full range of cancer types and as low as 16 nM in individual cell lines. A second in vitro screen in collaboration with researchers in the University of Nantes identified derivatives which could potently inhibit growth in a p53 mutant NSCLC cell line. The cell cycle effects of a selected panel of isoellipticines were studied in leukaemia cell lines by researchers in the Department of Biochemistry and Cell Biology, UCC. Emerging from this, the therapeutic potential of one of the derivatives in AML was then assessed in vivo in an AML xenograft mouse model, with tumour weight reduced by a factor of 7 in treated mice relative to control.