Christian Bailly

Cytotoxicity and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis

Cryptolepine and neocryptolepine are two indoloquinoline derivatives isolated from the roots of the african plant Cryptolepis sanguinolenta. These two alkaloids, which only differ by the respective orientation of their indole and quinoline rings, display potent cytotoxic activities against tumour cells and present antibacterial and antiparasitic properties. Our previous molecular studies indicated that these two natural products intercalate into DNA and interfere with the catalytic activity of human topoisomerase II. Here we have extended the study of their mechanism of action at the cellular level. Murine and human leukemia cells were used to evaluate the cytotoxicity of the drugs and their effects on the cell cycle were measured by flow cytometry. Cryptolepine, and to a lesser extent neocryptolepine, provoke a massive accumulation of P388 murine leukemia cells in the G2/M phase. With HL-60 human leukemia cells, the treatment with cryptolepine leads to the appearance of a hypo-diploid DNA content peak (sub-G1) characteristic of the apoptotic cell population. With both P388 and HL-60 cells, cryptolepine proved about four times more toxic than its isomer. But the use of the HL-60/MX2 cell line resistant to the anticancer drug mitoxantrone suggests that topoisomerase II may not represent the essential cellular target for the alkaloids, which are both only two times less toxic to the resistant HL-60/MX2 cells compared to the parental cells. The capacity of the drugs to induce apoptosis of HL-60 human leukemia cells was examined by complementary biochemical techniques. Western blotting analysis revealed that cryptolepine, but not neocryptolepine, induces cleavage of poly(ADP-ribose) polymerase but both alkaloids induce the release of cytochrome c from the mitochondria. The cleavage of poly(ADP-ribose) polymerase observed with cryptolepine correlates with the appearance of a marked sub-G1 peak in the cell cycle experiments. The proteolytic activity of Asp-Glu-Val-Asp- or Ile-Glu-Thr-Asp-caspases was found to be enhanced much more strongly with cryptolepine than with its isomer, as expected from their different cytotoxic potential. Despite the activation of the caspase cascade, we did not detect internucleosomal cleavage of DNA in the HL-60 cells treated with the alkaloids. Altogether, the results shed light on the mechanism of action of these two plant alkaloids.

Characterization of a Novel DNA Minor-Groove Complex

Many dicationic amidine compounds bind in the DNA minor groove and have excellent biological activity against a range of infectious diseases. Para-substituted aromatic diamidines such as furamidine, which is currently being tested against trypanosomiasis in humans, and berenil, which is used in animals, are typical examples of this class. Recently, a meta-substituted diamidine, CGP 40215A, has been found to have excellent antitrypanosomal activity. The compound has a linear, conjugated linking group that can be protonated under physiological conditions when the compound interacts with DNA. Structural and molecular dynamics analysis of the DNA complex indicated an unusual AT-specific complex that involved water-mediated H-bonds between one amidine of the compound and DNA bases at the floor of the minor groove. To investigate this unique system in more detail DNase I footprinting, surface plasmon resonance biosensor techniques, linear dichroism, circular dichroism, ultraviolet-visible spectroscopy, and additional molecular dynamics simulations have been conducted. Spectrophotometric titrations of CGP 40215A binding to poly(dAT)(2) have characteristics of DNA-binding-induced spectral changes as well as effects due to binding-induced protonation of the compound linker. Both footprinting and surface plasmon resonance results show that this compound has a high affinity for AT-rich sequences of DNA but very weak binding to GC sequences. The dissociation kinetics of the CGP 40215A-DNA complex are much slower than with similar diamidines such as berenil. The linear dichroism results support a minor-groove complex for the compound in AT DNA sequences. Molecular dynamics studies complement the structural analysis and provide a clear picture of the importance of water in mediating the dynamic interactions between the ligand and the DNA bases in the minor groove.

Tight binding of the antitumor drug ditercalinium to quadruplex DNA

The structural selectivity of the DNA‐binding antitumor drug ditercalinium was investigated by competition dialysis with a series of nineteen different DNA substrates. The 7H‐pyridocarbazole dimer was found to bind to double‐stranded DNA with a preference for GC‐rich species but can in addition form stable complexes with triplex and quadruplex structures. The preferential interaction of the drug with four‐stranded DNA structures was independently confirmed by electrospray mass spectrometry and a detailed analysis of the binding reaction was performed by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR study showed that the kinetic parameters for the interaction of ditercalinium with the human telomeric quadruplex sequence are comparable to those measured with a duplex sequence. Slow association and dissociation were observed with both the quadruplex and duplex structures. The newly discovered preferential binding of ditercalinium to the antiparallel quadruplex sequence d(AG3[T2AG3]3) provides new perspectives for the design of drugs that can bind to human telomeres.

Electric linear dichroism as a new tool to study sequence preference in drug binding to DNA

An original approach using electric linear dichroism (ELD) and natural DNAs and polynucleotides of differing base composition has been developed with the aim to investigate the sequence-dependent recognition of DNA by drugs. Both intercalators and minor groove binders have been studied as well as certain hybrid molecules. The results indicate that the orientation of drugs upon binding to nucleic acids can change markedly according to the target sequence. Among the intercalators tested, only actinomycin D and hycanthone show a clear preference for GC- and AT-rich sequences, respectively. For minor groove binders, the linear dichroism showing a strong dependence on base composition of the DNA and polynucleotides is most pronounced. Netropsin and distamycin bind to DNA with a marked AT specificity. Hoechst 33258, berenil and DAPI exhibit positive and negative dichroism signals at AT and GC sites respectively, suggesting that at least two types of drug-DNA interaction are involved depending on the AT/GC content of the DNA. Further investigations using polynucleotides with inosine substituted for guanosine, and competition experiments with intercalative drugs suggest that Hoechst 33258, berenil and DAPI interact with GC sequences via a non-classical intercalation process.

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

Exploring the Cellular Activity of Camptothecin-Triple-Helix-Forming Oligonucleotide Conjugates

ABSTRACT Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

Synthesis, biological evaluation and DNA binding properties of novel mono and bisnaphthalimides

A novel series of mono and bisnaphthalimides was synthesized and their antiproliferative activities were evaluated against three tumor cell lines. Bisnaphthalimides 3 and 4, bearing a pyrazine ring fused to the naphthalimide system, showed activities in the order of 10(-8) microM, similar to elinafide. DNA binding properties and the ability to induce DNA damage were studied for some of the most active compounds.

Synthesis and pharmacological evaluation of novel non-lactone analogues of camptothecin.

Ten novel camptothecin (CPT) derivatives devoid of the lactone function in the E-ring were synthesized and evaluated as anticancer agents. Several of these CPT analogues bearing a five-membered E-ring are potent inhibitors of the DNA relaxation and cleavage reactions catalyzed by topoisomerase I and exhibit promising cytotoxic activities with IC(50) values in the nM range. This is the first successful design of lactone-free CPT, providing thus a new avenue to the development of topoisomerase I targeted antitumor agents.

Synthesis of 3,5-bis(2-indolyl)pyridine and 3-((2-indolyl)-5-phenyl)- pyridine derivatives as CDK inhibitors and cytotoxic agents

We here report the synthesis and biological evaluation of new 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine designed as potential CDK inhibitors. Indole, 5-hydroxyindole, and phenol derivatives were used to generate three substitutions of the pyridine. The resulting skeletons were successively exploited to introduce various dimethylaminoalkyl side chains by Williamson type reactions. The synthesis includes Stille or Suzuki type reactions, which were realized on the 3,5-dibromopyridine. The preparation and the use of stannylindoles in mono or bis cross-coupling reactions were also described and each step was optimized and detailed. Kinase assays were realized and shown that nude compounds 7, 18, and 25 inhibited CDK1 in the 0.3-0.7 micromolar range with a good selectivity over GSK-3. Cytotoxicity against CEM human leukemia cells was evaluated with IC(50) values in the 5-15 micromolar range. Precise structure-activity relationships were delineated. Molecular modeling and docking solutions were proposed to complete the studies and to explain the observed SAR in the CDK assays.

Relation between intracellular acidification and camptothecin-induced apoptosis in leukemia cells.

Leukemia cells (HL-60 and P388) treated with the topoisomerase I inhibitor camptothecin (CPT) undergo rapid apoptosis as judged from internucleosomal degradation of genomic DNA, morphological changes and flow cytometry analysis. The intracellular free calcium concentration is not affected by the treatment with a high dose of CPT. In contrast, fluorescence measurements of cells loaded with the pH indicator BCECF-AM indicate that the intracellular pH decreases significantly. Incubation of the leukemia cells with a high drug concentration for 5 h or with lower drug concentrations for 15 h results in a pronounced intracellular acidification. Measurements with the whole cell population show a decrease of 0.3-0.4 pH units. The extent of the acidic shift is proportional to the drug concentration and the period of incubation. No such effects were observed with P388CPT5 cells resistant to CPT. The results support the hypothesis that apoptosis induced in leukemia cells by CPT is associated with decreased intracellular pH. Modification of intracellular pH by topoisomerase inhibitors is viewed as an essential event responsible for the induction and/or propagation of apoptosis. The role of CPT-induced cellular acidification in the mechanism of action of the drug is discussed.