Cellular effects of a new farnesyltransferase inhibitor, RPR-115135, in a human isogenic colon cancer cell line model system HCT-116.
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Alkyl and Aryl Transferases↗
Biomedical subjects
J F Riou
Publications and source records attributed to J F Riou.
Enhanced binding to DNA and topoisomerase I inhibition by an analog of the antitumor antibiotic rebeccamycin containing an amino sugar residue.
Many antitumor agents contain a carbohydrate side chain appended to a DNA-intercalating chromophore. This is the case with anthracyclines such as daunomycin and also with indolocarbazoles including the antibiotic rebeccamycin and its tumor active analog, NB506. In each case, the glycoside residue plays a significant role in the interaction of the drug with the DNA double helix. In this study we show that the DNA-binding affinity and sequence selectivity of a rebeccamycin derivative can be enhanced by replacing the glucose residue with a 2'-aminoglucose moiety. The drug-DNA interactions were studied by thermal denaturation, fluorescence, and footprinting experiments. The thermodynamic parameters indicate that the newly introduced amino group on the glycoside residue significantly enhanced binding to DNA by increasing the contribution of the polyelectrolyte effect to the binding free energy, but does not appear to participate in any specific molecular contacts. The energetic contribution of the amino group of the rebeccamycin analog was found to be weaker than that of the sugar amino group of daunomycin, possibly because the indolocarbazole derivative is only partially charged at neutral pH. Topoisomerase I-mediated DNA cleavage studies reveal that the OH-->NH2 substitution does not affect the capacity of the drug to stabilize enzyme-DNA covalent complexes. Cytotoxicity studies with P388 leukemia cells sensitive or resistant to camptothecin suggest that topoisomerase I represents a privileged intracellular target for the studied compounds. The role of the sugar amino group is discussed. The study provides useful guidelines for the development of a new generation of indolocarbazole-based antitumor agents.
Amino Sugars↗
The development of telomerase inhibitors: the G-quartet approach.
Human telomeres, which consist of repeated TTAGGG sequences, have recently become the focus of intense and highly competitive biological research. This scientific interest lies in their unique biological functions: telomeres are essential for genome integrity and appear to play an important role in cellular aging and cancer. As telomerase appears to be selectively expressed in tumors versus normal cells, this enzyme represents a good target for inhibition. Different types of telomerase inhibitors have recently been described. We will present briefly the different strategies that have been proposed to achieve efficient telomerase inhibition, with a special emphasis on G-quartet ligands.
Enzyme Inhibitors↗
Modulation in kinetics of lactone ring hydrolysis of camptothecins upon interaction with topoisomerase I cleavage sites on DNA.
The kinetics of hydrolysis of the alpha-hydroxylactone ring of anticancer agents belonging to the camptothecin (CPT) series has been followed using their fluorescence emission. Data obtained for CPT, CPT-11, and SN-38, either in their free form or in the presence of DNA and/or topoisomerase I (top1), have been compared. DNA was modeled using three types of double-strand oligonucleotides corresponding to top1 cleavage site enhanced in the presence of the drug (olg1), top1 site independent of CPT (olg2), and nonspecific synthetic oligonucleotide containing only AT and no GC base pairs (olg3). Cleavage assays indicated the absence of top1-mediated cleavage on olg3, both in the presence and in the absence of CPT. The kinetics data also showed ratio-dependent stabilization of the lactone forms of CPTs when in the presence of an excess of olg1 or olg2, but not of olg3. These observations correlate with the previously reported preferential binding of CPTs to guanines. Although lactone hydrolysis was not perturbed by top1 alone, this enzyme hindered lactone stabilization by specific oligonucleotides. After addition of top1 to CPT-olg1 or CPT-olg2 complexes, the lactone ring of the drug was destabilized. No lactone stabilization was observed when olg1 was added to CPT-top1 complexes or when olg1-top1 complexes were added to CPT.
Camptothecin↗
Syntheses and biological evaluation of indolocarbazoles, analogues of rebeccamycin, modified at the imide heterocycle.
A series of 10 indolocarbazole derivatives, analogues to the antitumor antibiotic rebeccamycin, bearing modifications at the imide heterocycle were synthesized. They bear an N-methyl imide, N-methyl amide, or anhydride function instead of the original imide. Their inhibitory potencies toward topoisomerase I were examined using a DNA relaxation assay and by analyzing the drug-induced cleavage of 32P-labeled DNA. Protein kinase C (PKC) inhibition and interaction with DNA were also studied together with the in vitro antiproliferative activities against B16 melanoma and P388 leukemia cells. The antimicrobial activities against two Gram-positive bacteria (Bacillus cereus and Streptomyces chartreusis), a Gram-negative bacterium (Escherichia coli), and a yeast (Candida albicans) were tested as well as their antiviral activities toward HIV-1. The efficiency of the anhydride compounds was compared to that of the parent compound rebeccamycin and its dechlorinated analogue. All the compounds studied were inactive against PKC. The structural requirements for PKC and topoisomerase I inhibition are markedly different. In sharp contrast with the structure-PKC inhibition relationships, we found that an anhydride function does not affect topoisomerase I inhibition, whereas a methyl group on the indole nitrogen prevents the poisoning of topoisomerase I. The compounds exhibiting a marked toxicity to P388 leukemia cells had little or no effect on the growth of P388CPT5 cells which are resistant to the topoisomerase I inhibitor camptothecin. This study reinforces the conclusion that the DNA-topoisomerase I cleavable complex is the primary cellular target of the indolocarbazoles and significantly contributes to their cytotoxicity and possibly to their weak but noticeable anti-HIV-1 activities. The structure-activity relationships are also discussed.
Aminoglycosides↗
Spectroscopic and biochemical characterisation of self-aggregates formed by antitumor drugs of the camptothecin family: their possible role in the unique mode of drug action.
We describe the effect strongly influencing the biological activity of some camptothecin (CPT) drugs, the inhibitors of DNA topoisomerase I (topo I), namely, the formation of J-type aggregates in an aqueous buffer solution. These aggregates were built up under certain dilution conditions of the stock DMSO solutions of 20-S-camptothecin (20(S)CPT), 10,11-methylenedioxy-CPT (10,11-CPT) and 7-ethyl-10-hydroxy-CPT (SN38). The aggregates were found to be stereospecific, not being detectable for the 20(R)-stereoisomer of CPT. They were formed by the stacking interaction between quinoline rings of CPT chromophores with the inverse position of the nitrogen atoms. The aggregates were stable at acidic and neutral pHs, but dissociated at basic pHs. Self-aggregation prevented hydrolysis of the lactone ring at neutral pHs, thus preserving the drugs in a biologically active form. Addition of BSA did not induce either disaggregation or hydrolysis of the lactone ring, whereas the monomeric form of the drugs was shown to undergo rapid conversion to an inactive carboxylate form in the presence of human serum albumin [5]. The drugs did not form the aggregates in the presence of topo I. Moreover, rapid dissociation of the aggregates was observed if a self-aggregated drug solution was added to topo I alone or to the DNA-topo I cleavage assay. Neither DNA alone nor oligonucleotides derived from the sequences of the CPT-enhanced or topo I-induced cleavage sites in SV40 plasmid DNA induces changes in the aggregation state of the drugs. These observations are indicative of interaction between the aggregates and topo I. The aggregates were found to penetrate within the cells with much higher efficiency than a monomeric form of the drugs. Cellular uptake of aggregated and nonaggregated species correlated well with cytotoxic effects produced by the drug. In this manner, CPT's self-aggregation should be regarded as a favourable phenomenon producing species with a more stable biologically active structure of the lactone ring and exhibiting enhanced cellular uptake levels relative to the monomeric forms of medications.
Antineoplastic Agents, Phytogenic↗
Kinetics of lactone hydrolysis in antitumor drugs of camptothecin series as studied by fluorescence spectroscopy.
Potent antitumor activity exhibited by 20-S-camptothecin (CPT) and numerous derivatives is known to be lost upon opening of the alpha-hydroxy-lactone ring of these drugs, hydrolyzable at neutral and basic pH. To quantify in 'real time' the lactone hydrolysis reaction in CPTs under physiological conditions, we have applied a non-perturbing approach by fluorescence spectroscopy. CPT and a set of its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38, topotecan, tricyclic ketone-CPT) with antitumor activity varying from negligible to 10 times that of CPT have been studied. Prior to the kinetic measurements, the effects of substitutions, pH, polarity of molecular environment, lactone ring opening (lactone-carboxylate transition) have been investigated in terms of the UV-visible absorption and fluorescence emission spectra of CPTs. Then the determined parameters of the fluorescence emission spectra corresponding to the respective lactone and carboxylate forms have been used to estimate the residual lactone percentage as a function of time. The reproducibility of the obtained data demonstrates that the spectroscopic approach provides a satisfactory precision for this kind of measurements. For CPT at pH 7.3, the lactone half-life was 29.4 +/- 1.7 min and the lactone percentage at equilibrium was 20.9 +/- 0.3%. Within a series of derivatives with substitutions at quinoline rings, the lactone half-life varied from 29 to 32 min and the equilibrium lactone content varied from 15% to 23%. For each compound, even slight increase of pH from 7.1 to 7.3 or from 7.3 to 7.6 logically leads to a remarkable decrease of both lactone half-life and equilibrium lactone percentage.
Antineoplastic Agents, Phytogenic↗
Role of fibroblast growth factor during early midbrain development in Xenopus.
Genes encoding fibroblast growth factors (FGFs) are expressed in early Xenopus neurulae in the prospective midbrain-hindbrain boundary (MHB) region of the neural plate. These expression domains overlap those of XWnt-1 and XEn-2, raising the question of the role of FGF signalling in the regulation of these genes, and more generally about the function of FGF during Xenopus midbrain development. We report that explants from the prospective MHB grafted into the anterior neural plate in midneurula stage embryos induce XWnt-1 expression and, at a lower frequency, XEn-2 expression in the vicinity of the graft. Such a process is likely to involve FGF signalling. Implantation of FGF4- or FGF8-soaked beads in the prospective forebrain at neurula and tailbud stages causes the up-regulation of XWnt-1 and XEn-2 in the dorsal and lateral region of the anterior midbrain. This effect is not relayed by endogenous FGF genes since exogenous FGFs inhibit the expression of endogenous XFGF3 or XFGF8. However, consequences of grafting MHB or implanting FGF4 or FGF8 beads on tadpole brain development are different. MHB grafts induce ectopic mesencephalic structures, strongly suggesting that a region homologous to the isthmic organizer of amniotes is specified as early as the midneurula stage. In contrast, exogenous FGFs do not cause the formation of ectopic mesencephalic structures but an overgrowth of mesencephalon and diencephalon. We propose that FGF signals from the prospective MHB play a crucial role in the spatial regulation of XWnt-1 and XEn-2 expression in the posterior midbrain, but that the full organizing activity of the MHB involves other factors in combination with FGF.
Animals↗
Syntheses, biochemical and biological evaluation of staurosporine analogues from the microbial metabolite rebeccamycin.
The indolocarbazole antibiotics staurosporine and rebeccamycin (1) are potent antitumor drugs targeting protein kinase C and topoisomerase I, respectively. To obtain staurosporine analogues from rebeccamycin, different structural modifications were performed: coupling of the sugar moiety to the second indole nitrogen, dechlorination and then reduction of the imide function to amide. The newly synthesized compounds (3-6) were tested for their abilities to bind to DNA and to inhibit topoisomerase I and protein kinase C. Their antiproliferative effects in vitro against B16 melanoma and P388 leukemia (including the related P388CPT cell line resistant to camptothecin) as well as their anti-HIV-1 and antimicrobial activities against various strains of microorganisms were determined. The cytotoxicity of the dechlorinated imide analogue 5 correlates well with its DNA binding and anti-topoisomerase I activities. These findings provide guidance for the development of new topoisomerase I-targeted antitumor indolocarbazoles equipped with a carbohydrate attached to the two indole nitrogens.
Aminoglycosides↗
Identification of a new metabolite of CPT-11 (irinotecan): pharmacological properties and activation to SN-38.
Irinotecan, or CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine++ +), is a water-soluble derivative of camptothecine with promising activity against several types of malignancies. In addition to 7-ethyl-10-hydroxycamptothecine (SN-38), its active metabolite, we were able to identify several metabolites in the plasma of patients treated with this drug, especially an oxidative metabolite, 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine. During our study of the biosynthesis of 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine from CPT-11 by human liver microsomes, we were able to detect another quantitatively important polar metabolite, which was also present in the plasma and urine of patients treated with CPT-11. On the basis of preliminary experiments, the structure of this compound was postulated to be 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine, and this structure was synthesized by Rhône-Poulenc Rorer. Urine samples and human liver microsomal extracts were studied by high-performance liquid chromatography/atmospheric pressure chemical ionization/tandem mass spectrometry to identify its structure formally. The identification of the metabolite was supported by identical retention time, mass-to-charge ratio and tandem mass spectrometry fragmentation as a synthetic standard. Like irinotecan, 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecine was a weak inhibitor of cell growth of P388 cells in culture (IC50 = 3.4 micrograms/ml vs. 2.8 micrograms/ml for irinotecan and 0.001 microgram/ml for SN-38). It was also a poor inducer of topoisomerase I-DNA cleavable complexes (100-fold less potent than SN-38). However, unlike 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine, this new metabolite could be hydrolyzed to SN-38 by human liver microsomes and purified human liver carboxylesterase, though to a lesser extent than irinotecan. This compound can therefore contribute to the activity and toxicity profile of irinotecan in vivo.
Animals↗
Synthesis, DNA binding, topoisomerase II inhibition and cytotoxicity of two guanidine-containing anthracene-9,10-diones.
Two anthraquinone derivatives of the anticancer drugs mitoxantrone and ametantrone were examined for their ability to bind to DNA and to modulate the formation of topoisomerase-DNA cleavable complexes in vitro. The guanidinium groups introduced at the termini of the two aminoethylamino side chains of mitoxantrone can reinforce the interaction with DNA as judged from thermal denaturation studies with calf thymus DNA and polynucleotides. Footprinting experiments indicate that the binding to DNA of compound SR107 lacking the 5,8-hydroxyl substituents is essentially nonspecific whereas its congener SR 103 interacts preferentially with GC-rich sequences, particularly those containing 5'-(A/T)CG sites. Compound SR103, which bears two hydroxyl groups on the anthraquinone chromophore, promotes the cleavage of DNA by topoisomerase II and is cytotoxic toward human KB carcinoma cells in vitro. In contrast, the analogue SR107, which lacks OH groups, has no effect on topoisomerase II and is not cytotoxic.
Anthraquinones↗
[in Vitro reconstitution of human telomerase activity].
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Animals↗
Another link between phospholipid transmembrane migration and ABC transporter gene family, inferred from a rare inherited disorder of phosphatidylserine externalization.
The mechanisms involved in the maintenance or loss of the asymmetric distribution of phospholipids in the cell plasma membrane remain mysterious. In the yeast Saccharomyces cerevisiae, the transmembrane migration of certain phospholipids is controlled by transcription regulators of various ATP-binding cassette (ABC) transporters. The P-glycoprotein membrane transporters encoded by the multidrug resistance (MDR) genes, members of the ABC protein family, act as lipid translocases in mammalian cells. We report here the lack of expression of MDR genes in lymphoblasts derived from the B cells of a patient with an inherited Scott syndrome, characterized by impaired transmembrane migration of procoagulant phosphatidylserine and hemorrhagic complications. From microsatellite analysis of 7q21.1 and functional assessment, the most likely explanation accounting for Scott phenotype is a mutation in an unlinked gene coding for a regulatory protein necessary for the expression of MDR genes. Because phosphatidylserine externalization is also one of the hallmarks of cells undergoing apoptosis, these observations are suggestive of a relationship between basic processes such as multidrug transport, apoptosis and procoagulant phospholipid exposure.
ATP Binding Cassette Transporter, Subfamily B↗
Syntheses and biological activities (topoisomerase inhibition and antitumor and antimicrobial properties) of rebeccamycin analogues bearing modified sugar moieties and substituted on the imide nitrogen with a methyl group.
As a part of studies on structure-activity relationships, several potential topoisomerase I inhibitors were prepared. Different analogues of the antitumor antibiotic rebeccamycin substituted on the imide nitrogen with a methyl group were synthesized. These compounds bore either the sugar residue of rebeccamycin, with or without the chlorine atoms on the indole moieties, or modified sugar residues (galactopyranosyl, glucopyranosyl, or fucopyranosyl) linked to the aglycone via a beta- or alpha-N-glycosidic bond. Their inhibitory properties toward protein kinase C, topoisomerase I, and topoisomerase II were examined, and their DNA-binding properties were investigated. Their in vitro antitumor activities against murine B16 melanoma and P388 leukemia cells were determined. Their antimicrobial activities were tested against Gram-positive bacteria Bacillus cereus and Streptomyces chartreusis, Gram-negative bacterium Escherichia coli, and yeast Candida albicans. These compounds are inactive toward topoisomerase II but inhibit topoisomerase I. A substitution with a methyl group on the imide nitrogen led to a loss of proteine kinase C inhibition in the maleimide indolocarbazole series but did not prevent topoisomerase I inhibition. Compounds possessing a beta-N-glycosidic bond, which fully intercalated into DNA, were more efficient at inhibiting topoisomerase I than their analogues with an alpha-N-glycosidic bond; however, both were equally toxic toward P388 leukemia cells. Dechlorinated rebeccamycin possessing a methyl group on the imide nitrogen was about 10 times more efficient in terms of cytotoxicity and inhibition of topoisomerase I than the natural metabolite.
Aminoglycosides↗
DNA cleavage by topoisomerase I in the presence of indolocarbazole derivatives of rebeccamycin.
DNA topoisomerase I has been shown to be an important therapeutic target in cancer chemotherapy for the camptothecins as well as for indolocarbazole antibiotics such as BE-13793C and its synthetic derivatives NB-506 and ED-110 [Yoshinari et al. (1993) Cancer Res. 53, 490-494]. To investigate the mechanism of topoisomerase I inhibition by indolocarbazoles, we have studied the induction of DNA cleavage by purified mammalian topoisomerase I mediated by the antitumor antibiotic rebeccamycin and a series of 20 indolocarbazole derivatives. The compounds tested bear (i) various functional groups on the non-indolic moiety (X = CO, CH2, CHOH), (ii) a hydrogen or a chlorine atom at positions 1 and 11 (R2), and (iii) different substituents on the maleimido function (R1 = H, OH, NH2, NHCHO). Half of the ligands have the same carbohydrate moiety as rebeccamycin whereas the other ligands have no sugar residue. The inhibitory potency of the test compounds was assessed in vitro by comparing the cleavage of [32P]-labeled restriction fragments by the enzyme in the absence and presence of the drug. In addition, the DNA-binding properties of these compounds were investigated by means of complementary spectroscopic techniques including electric linear dichroism, and the DNA sequence selectivity was probed by DNase I footprinting. The study shows that the sugar residue attached to the indolocarbazole chromophore is critical for the drug ability to interfere with topoisomerase I as well as for the formation of intercalation complexes. Structure-activity relationships indicate that the presence of chlorine atoms significantly reduces the effects on topoisomerase I whereas the substituents on the maleimido function and the functional group on the non-indolic moiety can be varied without reduction of activity. The results suggest that the inhibition of topoisomerase I by indolocarbazoles arises in part from their ability to interact with DNA. Analysis of the base preferences around topoisomerase I cleavage sites in various restriction fragments indicated that, in a manner similar to camptothecin, the rebeccamycin analogue R-3 stabilized topoisomerase I preferentially at sites having a T and a G on the 5' and 3' sides of the cleaved bond, respectively. By analogy with models previously proposed for camptothecin and numerous topoisomerase II inhibitors which intercalate into DNA, a stacking model for the interaction between DNA, topoisomerase I and indolocarbazoles is proposed. These findings provide guidance for the development of new topoisomerase I-targeted antitumor indolocarbazole derivatives.
Aminoglycosides↗
Structure-activity relation in camptothecin antitumor drugs: why a detailed molecular characterisation of their lactone and carboxylate forms by Raman and SERS spectroscopies?
Lactone and carboxylate forms of potent antitumor agents, camptothecins (CPTs) have been studied by Raman, Fourier-transform Raman (FT-Raman) and surface-enhanced Raman scattering (SERS) spectroscopy. Similarity of the Raman spectra of CPTs with corresponding FT-Raman spectra in the near-infrared allowed the latter to be compared with their SERS counterparts in order to analyse the interaction of the drugs with silver colloids. Different types of silver colloids (reduced with sodium borohydride or sodium citrate, with or without activation by anions) have been evaluated. Citrate-reduced colloid, activated with chloride anions (CAS) has been found to be the best compromise for SERS studies of both forms of CPTs. We suggest that in general CPTs are adsorbed on CAS via the nitrogen in ring B and are more inclined to a flat orientation than to a perpendicular one. However, probable interactions of substitution groups and/or of the COO- groups of hydrolysed CPTs with the CAS surface introduce some particularities in the adsorption patterns. As a result, SERS spectra are highly sensitive to hydrolysis and substitutions at distant rings of CPT and uniquely characteristic of each of the CPT derivatives. The pronounced hydrolysis-induced changes, similar in the Raman and SERS spectra of CPTs, involve similar vibrations in the spectra of different CPTs. Vibrational assignments, proposed for the main Raman and SERS bands of CPT and its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38 and topotecan) indicate that most of the bands which decreased upon lactone hydrolysis are those preferentially related to stretching modes of the quinoline rings A and B, and the bands which increased are those of the ring D stretching modes. Our data make the spectroscopic approach very promising for the further investigations of the molecular mechanisms of biological activity of CPTs.
Antineoplastic Agents↗
A carbamate analogue of amsacrine with activity against non-cycling cells stimulates topoisomerase II cleavage at DNA sites distinct from those of amsacrine.
AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta.
Amsacrine↗
Synthesis and biological evaluation of monoindolyl and indolocarbazolyl oxazolones and imidazolones.
Eight compounds structurally related to protein kinase C inhibitor MDL 27032 and substituted with indole moieties were synthesized. Their activities towards protein kinase C (PKC) and protein kinase A (PKA) were determined. Their effect on PKC-mediated contraction of rat tracheal smooth muscle, their antiproliferative activity on two murine tumor cell lines, melanoma B16 and leukemia P388 and their antimicrobial activity on a gram-positive bacterium Bacillus cereus were also examined. The mammalian and bacterial cell antiproliferative activity, as well as vasorelaxant effect, observed for some of them could not be correlated to PKC or PKA inhibition. Only bulky bis-indolyl compounds exhibited biological activity in these experiments. Rigid indolocarbazoles had the strongest antiproliferative activity.
Animals↗