Biomedical subjects

C Garbay-Jaureguiberry

Publications and source records attributed to C Garbay-Jaureguiberry.

At least 19 recordsLinked to original sources

PubMed2000

Effects of cationic charge on three-dimensional structures of intercalative complexes: structure of a bis-intercalated DNA complex solved by MAD phasing.

We characterize intercalative complexes as either "high charge" and "low charge". In low charge complexes, stacking interactions appear to dominate stability and structure. The dominance of stacking is evident in structures of daunomycin, nogalamycin, ethidium, and triostin A/echinomycin. By contrast in a DNA complex with the tetracationic metalloporphyrin CuTMPyP4 [copper (II) meso-tetra(N-methyl-4-pyridyl)porphyrin], electrostatic interactions appear to draw the porphyrin into the duplex interior, extending the DNA along its axis, and unstacking the DNA. Similarly, DNA complexes of tetracationic ditercalinium and tetracationic flexi-di show significant unstacking. Here we report x-ray structures of complexes of the tetracationic bis-intercalator D232 bound to DNA fragments d(CGTACG) and d(BrCGTABrCG). D232 is analogous to ditercalinium but with three methylene groups inserted between the piperidinium groups. The extension of the D232 linker allows it to sandwich four base pairs rather than two. In comparison to CuTMPyP4, flexi-di and ditercalinium, stacking interactions of D232 are significantly improved. We conclude that it is not sufficient to characterize intercalators simply by net charge. One anticipates strong electrostatic forces when cationic charge is focused to a small volume or region near DNA and so must consider the extent to which cationic charge is focused or distributed. In sum, ditercalinium, with a relatively short linker, focuses cationic charge more narrowly than does D232. So even though the net charges are equivalent, electrostatic charges are expected to be of greater structural significance in the ditercalinium complex than in the D232 complex.

Cations↗

PubMed1995

Asymmetry and dynamics in bis-intercalated DNA.

The bis-intercalator ditercalinium (NSC 366241), composed of two 7 H-pyridocarbazoles linked by a bis(ethylpiperidinium), binds to DNA with a binding constant greater than 10(7) M-1. One distinctive aspect of the 3-D X-ray structure of a DNA-ditercalinium complex is its asymmetry. We propose here that the activity of ditercalinium may be related to structural polymorphism and dynamic conversion between conformers. It was previously reported that activity is closely related to linker composition. Activity increases with increasing conformational restraints of the linker. We suggest these conformational restraints can lead to asymmetry in DNA complexes and that this asymmetry results directly in structural polymorphism. Using the Cambridge Structural Database (CSD) as a source of information about chemical fragments that are analogous to the linker of ditercalinium, we have explored the conformational space available to ditercalinium. The results indicate that the linker is highly constrained and that the DNA complex is intrinsically asymmetric. We propose a reasonable mechanism of ring reversal that is consistent with the conformations of analogous fragments within the CSD.

Antineoplastic Agents↗

PubMed1994

DNA distortion in bis-intercalated complexes.

The bis-intercalators Flexi-Di and ditercalinium are synthetic dimers that bis-intercalate into DNA and cause cell death in prokaryotes from futile and abortive repair of DNA. Each is composed of two 7H-pyridocarbazole units and a linker. Flexi-Di has a flexible spermine-like linker while ditercalinium has a rigid bis(ethylpiperidinium) linker. This report, describing the 2.5-A X-ray structure of Flexi-Di complexed with [d(BrCGCG)]2, appears to be the first report of a three-dimensional structure of a DNA complex with a bis-intercalator with a flexible linker. DNA complex formation with a ditercalinium analog having a flexible linker was not anticipated to yield unstacked and bent DNA as was observed in the previously reported ditercalinium.[d(CGCG)]2 complex. Surprisingly, the DNA in the Flexi-Di complex is bent to a degree exceeding that of the ditercalinium complex. A comparison of the DNA complexes of Flexi-Di and ditercalinium has allowed us to propose a mechanism by which these bis-intercalators distort DNA. We propose that this class of bis-intercalators pulls the internal base pairs into the major groove and pushes the external base pairs into the minor groove. The result is a bend toward the minor groove. It appears that hydrogen bonds between the linker and the internal guanines effectively pull the central base pairs of the complex out into the major groove. At the external regions of the complex, stacking interactions between the chromophores and terminal base pairs effectively push the terminal base pairs into the minor groove. The result of this push/pull combination is to bend the DNA.

Base Sequence↗

PubMed1994

Fate of a fluorescent inhibitor of endopeptidase-24.11 using enzyme-expressing MDCK cells. Modification of its cellular processing with a monoclonal antibody.

Neutral endopeptidase-24.11 (NEP) is a membrane-bound zinc metallopeptidase which cleaves biologically active peptides such as the enkephalins and atrial natriuretic peptide. Using the specific and fluorescent thiol inhibitor of the enzyme, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl)-amino-1- hexyl]-thiocarbamide (FTI), the fate of the inhibitor-enzyme complex was investigated by videomicrofluorimetry using MDCK epithelial cells expressing the rabbit peptidase thanks to a retroviral expression vector. N-[3-(R,S)-[(hydroxyamino) carbonyl]-2-benzyl-1-oxopropyl]- glycine (HACBOGly) and the corresponding tritiated molecule were also used to measure the cellular pathway of inhibitor-NEP complexes. In the present paper, we demonstrate that, for short incubation times, the fluorescent probe preferentially labeled brush border membranes of the apical side of the MDCK cells. After more than 1 h incubation, a honeycomb pattern of fluorescence was observed in videomicrofluorimetry suggesting that part of the inhibitor was bound or localized close to the basolateral plasma membrane. Confocal experiments confirmed the transcytosis of FTI/NEP complex, from the apical to the basolateral domain. Using [3H]HACBOGly on filter-grown cells, after 2 and 4 h incubation at 37 degrees C, the percentage of basolateral membrane-bound molecules was estimated to be about 12 and 23%, respectively. The coincubation of the cells with FTI and 2B12, a monoclonal antibody raised against the rabbit enzyme, greatly modified the fluorescence pattern. A patchy fluorescence was observed for short incubation times, corresponding to cluster formation induced by antigen-antibody binding. For longer incubation times (> 1 h), in addition to the basolateral labeling, some intracellular fluorescent vesicles were observed essentially localized in the vicinity of the nucleus. The colocalization of FTI with Texas Red isothiocyanate-labeled Concanavalin A (TRITC-Con A) strongly suggests an endosomal/lysosomal internalization pathway when FTI was incubated in the presence of 2B12 mAb.

Animals↗

PubMed1993

Synthesis and structure-activity studies of a series of [(hydroxybenzyl)amino]salicylates as inhibitors of EGF receptor-associated tyrosine kinase activity.

The synthesis and structure-activity relationships of a series of [(hydroxybenzylidene)amino]salicylates and a series of [(hydroxybenzyl)amino]salicylates as inhibitors of EGF receptor-associated tyrosine kinase activity are described. Their inhibitory potency was evaluated in vitro using ER 22 cell membranes (CCL 39 cells transfected with EGF receptor) as an enzyme source and the tridecapeptide RRSrc (RRLIEDAEYAARG) as substrate. Their cellular activity was measured by inhibition of the EGF-stimulated DNA synthesis of ER 22 cells. Chemical modifications were made to analyze the role of the different substituents. The amino series was found to be more active than the imino series. The hydroquinone moiety appears to be essential for tyrosine kinase inhibitory activity in the series of 5-[(2,5-dihydroxybenzyl)amino]salicylates. Comparison of the imino and amino series by molecular modeling techniques provides further evidence in support of the hypothesis that the important reduced linking chain, CH2NH, allows the correct positioning of the 2,5-dihydroxybenzyl ring, possibly in a cis-like conformational arrangement.

Aminosalicylic Acids↗

PubMed1993

[125I]azido-DTLET as a tool for selective covalent labeling of delta-opioid receptors in rat brain sections.

The binding kinetics and pharmacological selectivity of the photoaffinity delta-opioid ligand [125I]azido-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr) were investigated in serial frozen sections from rat neostriatum prior to ultraviolet irradiation (i.e., in conditions of reversibility). Scatchard analysis of saturation binding experiments indicated that [125I]azido-DTLET binds to both a high (KD = 5.04 nM) and a low (KD = 38 nM) affinity site. Binding to the low-affinity site was no longer detectable in the presence of unlabeled [D-Ala2,N-MePhe4,Gly-Ol5]enkephalin (DAGO), suggesting that this site corresponds to mu-opioid receptors. This interpretation was further supported by the dose-dependent inhibition of the binding of [3H]DAGO by non-radioactive azido-DTLET. Binding to the high-affinity site was totally inhibited, in a dose-dependent fashion, by a variety of opioid drugs among which delta-opioid ligands showed the highest order of potency. It is concluded that, in the nanomolar range, [125I]azido-DTLET constitutes a highly selective tool for covalent labeling of delta-opioid receptors in rat brain sections.

Affinity Labels↗

PubMed1992

Electron microscopic localization of photoaffinity-labelled delta opioid receptors in the neostriatum of the rat.

The distribution of delta opioid receptors, selectively labelled in vitro with the photoaffinity probe monoiodo azido-DTLET ([D-Thr2,pN3Phe4, Leu5]enkephaly-Thr6), was analyzed by light and electron microscopic radioautography in sections from rat neostriatum. Preliminary experiments indicated that up to 65% of specific 125I-azido-DTLET binding to rat striatal sections was still detectable following prefixation of the brain with 0.5% glutaraldehyde. These experiments also showed that up to 20-30% of the specifically bound radioactivity was covalently linked following ultraviolet irradiation and was thereby retained in tissue during subsequent postfixation and dehydration steps. Accordingly, the topographic distribution of the covalently attached azido-DTLET molecules was similar to that seen in fresh frozen sections and characteristic of that previously described for delta sites. Light and electron microscopic examination of the label in prefixed, striatal sections irradiated with ultraviolet light revealed that a significant proportion of specifically bound 125I-azido-DTLET molecules was intraneuronal. Specifically, 16% of the labelled binding sites were found in dendrites, 12% in perikarya and 4% in axon terminals. These results suggest that an important proportion of delta opioid binding sites labelled in the neostriatum correspond to receptors that are undergoing synthesis, transport and/or recycling. They also imply that a major fraction of delta sites are associated with intrastriatal neurons, as opposed to afferent axons. Approximately 44% of the labelled binding sites were associated with neuronal plasma membranes. Although most of these were found at the level of axodendritic (20%) and dendrodendritic (7%) appositions, comparison of the labelling incidence of these two compartments with their frequency of occurrence in tissue suggested that delta sites are fairly widely dispersed along neuronal plasma membranes. Only a small proportion (smaller than that of mu or kappa sites labelled in the same region) was associated with synaptic specializations. These results support the concept that delta receptors correspond to molecular entities that are distinct from mu and kappa sites and suggest that delta ligands act primarily nonjunctionally on the plasma membrane of striatal neurons.

Affinity Labels↗

PubMed1992

Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor.

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.

Aminopeptidases↗

PubMed1992

Increase of neutral endopeptidase-24.11 with cellular density and enzyme modulation with an inhibitor on human Reh6 cell line.

Neutral endopeptidase (EC 3.4.24.11, NEP) is an ectoenzyme, identified as the common acute lymphoblastic leukemia antigen (CALLA, CD10). This enzyme is involved in the inactivation of regulatory peptides such as enkephalins and atrial natriuretic peptide and its expression on the cell surface is therefore essential. NEP levels have been measured under different conditions on leukemic cell lines. NEP activity per cell was found to increase during the cell growth of Reh6 and CEM cells, a cell-cell contact mechanism being suggested by experiments using Transwell cell chambers. The same process was not observed with ICIG-7 fibroblasts. The numbers of enzymatic sites was also found to be selectively modulated by treatment with 0.1 microM N-[3-(R,S)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]glycine (HACBOGly), a potent (Ki = 1.4 nM) and specific inhibitor of NEP. A maximal 13% decrease in sites was observed after 8 hr incubation, this effect disappearing after 12 hr. This weak but specific negative modulation was not observed with a compound, chemically related to HACBOGly, which has a 10,000-fold lower inhibitory potency. The modulation was inhibited by low temperature or monensin treatment and could be brought about by an internalization of the enzyme, compensated for by an increased biosynthesis or by the sequestration of NEP in a non-membranous compartment.

Antigens, Differentiation↗

PubMed1992

Synthesis and biological activity of new dimers in the 7H-pyrido[4,3-c] carbazole antitumor series.

Ditercalinium (NSC 366241) is a 7H-pyrido[4,3-c]carbazole dimer with a diethylbipiperidine rigid chain linking the two heterocyclic rings. Ditercalinium is characterized by a high DNA affinity and bisintercalating ability, associated with potent antitumor properties, involving an original mechanism of action. Unfortunately as ditercalinium is hepatotoxic, its clinical evaluation has been interrupted. In order to eliminate or at least minimize the serious drawbacks related to its toxic effects, several chemical modifications have been made to the structure of ditercalinium, and their influence has been evaluated by measuring the DNA affinities, intercalation properties, and toxicity toward leukemia cells of the newly synthesized dimers. Reduction of the pyridinic moieties of ditercalinium, in order to suppress the permanent charges provided by the quaternizing chain, led to an almost complete loss of activity, although the DNA bisintercalating property of the dimer was preserved. Dimerization of the 7H-pyrido[4,3-c]carbazole rings by introduction of the rigid spacer on the N7- or C6-positions corresponding to the convex face of the pyridocarbazole, instead of the N2-position in ditercalinium, led to DNA bisintercalating dimers practically devoid of antitumor properties. However after quaternarization of the N2 atoms, the dimer linked by the N7 atoms exhibited a very high DNA affinity (greater than 10(9) M-1) and recovered antitumor activity, supporting the requirement of positive charges for the emergence of antitumor activity in these dimers. Introduction on the C6 of the 7H-pyridocarbazole ring of an aminomethyl or carboxyl group, a sugar residue, or C or N free amino acids such as Lys or Glu has also been carried out, in order to increase the hydrophilic properties of the molecules or to enable them to use amino acid transport systems. Although some of these compounds were active, none of them exhibited the pharmacological potency of ditercalinium.

Animals↗

PubMed1992

Solid phase synthesis of peptides containing the non-hydrolysable analog of (O)phosphotyrosine, p(CH2PO3H2)Phe. Application to the synthesis of 344-357 sequences of the beta 2 adrenergic receptor.

Studies about phosphorylation-dephosphorylation mechanisms require the development of probes capable of being used in in vitro and in vivo conditions. We show in this work that the chemically and enzymatically stable p(CH2PO3H2)Phe analog of (O)phosphotyrosine can be easily introduced in peptides by the solid-phase method. It has been incorporated in the 344-357 sequence of the beta 2 adrenergic receptor in place of the Tyr residue in position 350 and/or 354 in order to investigate the role of tyrosine phosphorylation in the receptor agonist-induced down-regulation. Since p(CH2PO3H2)Phe is an ionized hydrophilic residue, peptides containing this amino acid do not easily permeate the cellular membranes. Therefore the modified amino acid was introduced in the synthetic pathway in its N-Boc-p(CH2PO3Et2)Phe form, which could be partially or completely deprotected. Coupling steps, including that of the new amino acid, were performed with good yields (approximately 60% total yield) and further deprotections provided both the p(CH2PO3H2)Phe and p(CH2PO3HEt)Phe containing peptides with yields of around 20% each. The structure of the peptides was assessed by NMR, mass spectroscopy and amino acid analysis and the new amino acid was characterized under its phenylthiocarbamyl form (PTC).

Amino Acid Sequence↗

PubMed1991

Comparison of the bis-intercalating complexes formed between either ditercalinium or a flexible analogue and d(CpGpCpG)2 or d(TpTpCpGpCpGpApA)2 minihelices: 1H- and 31P-NMR analyses.

The 400-MHz 1H- and 162-MHz 31P-nmr have been used to study complexes constituted by (a) the d(TpTpCpGpCpGpApA)2 or the d(CpGpCpG)2 self-complementary oligonucleotides and (b) two bifunctional 7H-pyrido [4,3-c] carbazole dimer drugs, the antitumoral ditercalinium (NSC 366241), a dimer with a rigid bis-piperidine linking chain and its pharmacologically inactive analogue, a dimer with a flexible spermine-like linking chain. Nearly all proton and phosphorus signals have been assigned by two-dimensional (2D) nmr (correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser enhancement spectroscopy, 2D 31P (1H) heteronuclear correlated spectroscopy and 31P-31P chemical exchange experiments). Both drugs bis-intercalate into the two CpG sites. The complexes show small differences in the position of the 7H-pyrido [4,3-c] carbazole ring into the intercalation site and possibly in the ribose-phosphate backbone deformation. However, the inactive analogue exhibits a longer residence lifetime in octanucleotide than the ditercalinium does. All these results are discussed in terms of differences in dimer activities.

Base Sequence↗

PubMed1989

1H and 31P nuclear magnetic resonance studies of the differences in DNA deformation induced by anti-tumoral 7H-pyrido[4,3-c]carbazole dimers.

Ditercalinium (2,2'-[( 4,4'-bipiperidine]-1,1'-diyldi-2,1-ethane-diyl) bis-[10-methoxy-7H pyrido[4,3-c]carbazolium)tetramethane sulfonate (NSC 366241], a DNA bis-intercalating compound, is a potent anti-tumoral rigid dimer. Previous studies have shown that a reduced flexibility of the linking chain of such a dimer is essential for its biological activity. In order to understand, at the molecular level, the mechanism of action and the structure-activity relationships of this series of DNA intercalators, new dimers with additional methylene groups between the two piperidine rings have been synthesized. Addition of one methylene group in the chain preserved the activity, whereas addition of two methylene groups reduced the cytotoxicity, which finally disappeared when three methylene groups were inserted. Therefore, the study of the interaction of dimers bearing no (202), two (222) and three (232) methylene groups with the self-complementary hexanucleotide d(CGATCG)2 have been investigated by 1H and 31P nuclear magnetic resonance studies. The results reported here indicate that all dimers bis-intercalate into the minihelix. The intermolecular nuclear Overhauser effects (NOEs) between the dimers and the nucleotide lead to the conclusion that the three dimers intercalate with their rigid bis-ethyl bipiperidine chain fitting the major groove of the helix. Inter-residue nuclear Overhauser effects at the DNA level, as well as induced shifts, are discussed in relation to the conformational changes induced in DNA upon intercalation and to the different activity of the dimers.

Antineoplastic Agents↗

PubMed1989

Attempts to target antitumor drugs toward opioid receptor-rich mouse tumor cells with enkephalin-ellipticinium conjugates.

Human colorectal and pulmonary carcinomas have been shown to contain high levels of opioid peptides and their corresponding membrane-bound receptors. Therefore possible targeted drugs, consisting of modified enkephalins linked to cytotoxic drugs, were designed. Such conjugates were expected to be specifically internalized within opioid receptor-bearing cells. As a model to this approach, we have synthesized enkephalin-ellipticinium conjugates in which the D-Ala2-D-Leu5-enkephalin (DADLE) was coupled to the 2-nitrogen of either ellipticine or 9-hydroxyellipticine, two drugs acting through different mechanisms of cytotoxicity. These conjugates, DADLE-ellipticinium (NME) and DA-DLE-9-hydroxyellipticinium (NMHE), respectively, were previously shown to retain in vitro both opioid receptors and DNA affinities close to those of the parent compounds. In this paper, we first show that each individual moiety in the complexes remains capable of recognizing its cellular targets. Thus, pretreatment of NG108-15 cells containing delta-opioid receptors by the DADLE-ellipticinium conjugates induced a loss of opioid receptor (down-regulation), while the smaller peptide conjugates, tyrosinyl-D-alanylglycine-ellipticinium, prepared as control, do not. On the other hand, peptide-NMHE conjugates were able to induce DNA topoisomerase II-associated DNA strand breaks suggesting that they have a mode of action similar to that of their parent molecule, NMHE. We then examined whether or not these molecules could exert a specific toxicity on opioid receptor-bearing cells. However, when tested on NG108-15 tumor cells and L-fibroblasts as control, the enkephalin-ellipticinium conjugates (DADLE-NME and DADLE-NMHE) proved to be similarly more cytotoxic on both cell lines than their ellipticinium (NME and NMHE) precursors. In order to understand this apparent lack of specificity we examined the cellular accumulation and distribution of DADLE-NME by fluorescence techniques. These experiments revealed that an important intracellular overconcentration caused by a nonspecific process is probably masking the specific targeted effect of the conjugates. Hence, the project of linking DADLE to highly cytotoxic molecules which cannot cross the plasma membrane without site-directed targeting is discussed.

Alkaloids↗

PubMed1989

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters.

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.

Acridines↗

PubMed1988

Asymmetrical bisintercalators as potential antitumor agents.

Ditercalinium and its analogues are dimeric molecules made up of two identical 7H-pyrido[4,3-c]carbazole rings linked by symmetrical linking chains. These dimers elicit antitumor properties through a new mechanism of action. Recently, a relationship was found between their antitumor properties and their cytotoxic effect on the polA Escherichia coli mutant strain, suggesting that 7H-pyrido[4,3-c]carbazole dimers might induce a DNA deformation that could be recognized by the E. coli SOS repair system. Thus, the role of symmetry in ditercalinium analogues for their DNA binding, antitumor properties, and bacterial toxicity is investigated in the present study, by introducing asymmetric parameters in their structures. Dimers were either synthesized with an asymmetrical rigid linking chain or made up of two chemically different chromophores, i.e., acridine and 7H-pyrido[4,3-c]carbazole. The asymmetrical dimers remain able to bisintercalate into DNA with high affinities, but a dramatic loss in their antitumor potency is observed. On the other hand, these asymmetrical dimers are cytotoxic for polA E. coli mutants, like their symmetrical analogues. These results show that the symmetry plays a crucial role for the antitumor potency in the 7H-pyrido[4,3-c]carbazole dimers series.

Animals↗

PubMed1988

Photoaffinity labeling of opioid delta receptors with an iodinated azido-ligand: [125I][D-Thr2,pN3Phe4,Leu5]enkephalyl-Thr6.

The photoaffinity ligand of the delta opioid receptor Tyr-D-Thr-Gly-pN3Phe-Leu-Thr (azido-DTLET) was iodinated and purified by high performance liquid chromatography. Monoiodo-azido-DTLET displayed a high affinity (KD = 15 nM) and is selective (Kl mu/Kl delta = 9.8) for rat brain delta opioid receptors (for comparison, the corresponding values for tritiated azido-DTLET are KD = 1.66 nM and Kl mu/Kl delta = 27). On rat brain sections, the anatomical distribution of [125I]azido-DTLET binding sites revealed by autoradiography corresponds to that of delta receptors. On rat brain membrane homogenates and NG108-15 hybrid cells, UV irradiation of the receptor-ligand complex results in the irreversible binding to membrane proteins of 14% of the bound radioactivity Gel electrophoresis of [125I]azido-DTLET-labeled proteins followed by autoradiography shows a different pattern in rat brain and NG108-15 cells. In rat brain, labeling of two of these proteins, with molecular weights of 44,000 and 34,000, was inhibited by 30 nmol/liter of nonradioactive DTLET, a delta-selective ligand but not by the same concentration of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, a mu-selective ligand. In NG108-15 cells, this 44-kDa protein was not visualized; the main band was at 33 kDa and disappeared in the presence of levorphanol.

Affinity Labels↗

PubMed1988

Relationship between the size and position of substituents on 7H-pyrido[4,3-c]carbazole monomers and dimers and their DNA binding and anti-tumor properties.

Among the various DNA intercalating molecules prepared in our laboratory, ditercalinium, made up of two 7H-pyrido[4,3-c]carbazole rings linked by a rigid bis-ethylbipiperidine chain (NSC 366241) displays high anti-tumor properties. This dimeric molecule elicits its cytotoxicity through an original mechanism of action. At the molecular level, interaction of ditercalinium with the self-complementary d(CpGpCpG)2 nucleotide has been studied by 1H n.m.r. and the geometry of the bis-intercalating complex has been elucidated. Ditercalinium bis-intercalates through the major groove with the convex face of the 7H-pyrido[4,3-c]carbazole ring oriented toward the sugar moiety. Previous studies have shown the strong modulating effect brought about by substitution of the intercalating ring by methyl groups on both the DNA binding and anti-tumor potency of 7H-pyridocarbazole monomers and dimers. In order to further investigate structure activity relationships in the ditercalinium and related monomers series, a new family of monomeric and dimeric 7H-pyrido[4,3-c]carbazoles was prepared with ethyl, isopropyl or hydroxyethyl substitution on position 6 or 7 of the aromatic ring. DNA binding and anti-tumor potency of these compounds are reported. In contrast to their methylated analogs, none of the 6- or 7-substituted monomers studied here are active although most of them remain able to intercalate into DNA. Substitution of dimeric molecules by substituents of increasing size induces a progressive decrease in the anti-tumor potency, concomitantly with a disappearance of bis-intercalative properties. The modulating effect of the size of substituents on the DNA binding and anti-tumor properties of dimers is more pronounced in the 7-substituted series. These results are discussed in relation to the geometry of intercalation of ditercalinium and its related monomer into d(CpGpCpG)2.

Antineoplastic Agents↗