Showing papers by "Yves Pommier published in 1997"

Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function

Abstract: 7-Hydroxystaurosporine (UCN-01) is a selective protein kinase C inhibitor in clinical trial for cancer treatment. In this study, we found that nanomolar concentrations of camptothecin (CPT), a topoisomerase I inhibitor, arrest or delay cell cycle progression during the S and G2 phases in p53 mutant human colon carcinoma HT29 cells and that UCN-01 abrogates the S-phase arrest or delay induced by CPT. Under these conditions, CPT increased cyclin A levels and cyclin A/cyclin-dependent kinase 2 activity. UCN-01 prevented the increase of cyclin A/cyclin-dependent kinase 2 activity induced by CPT and enhanced Cdc2 kinase activity. Replication protein A (RPA2) was hyperphosphorylated after CPT treatment, and this effect was also abrogated by UCN-01. UCN-01 potentiated the cytotoxicity of CPT and reduced by 6-fold the concentration of CPT required to kill 50% of the HT-29 cells, as determined by clonogenic assays. This effect was observed at concentrations of UCN-01 that alone were not cytotoxic and had no detectable effect on cell cycle progression. UCN-01 markedly potentiated the cytotoxicity of CPT also in HCT116/E6 and MCF-7/ADR cells defective for p53 function, whereas significantly less potentiation was observed in p53-wild-type HCT116 and MCF-7 cells. These results suggest the existence of an S-phase checkpoint that delays replication and that may extend the time available for DNA repair. Thus, pharmacological abrogation of CPT-induced S- and G2-phase checkpoints by UCN-01 may provide an effective strategy for enhancing the chemotherapeutic activity of CPT, particularly against p53-defective tumors.

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

Abstract: Seventeen lichen acids comprising depsides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200 000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 μM; 15 were below 50 μM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

Abstract: Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN), caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200 000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3‘-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on t...

Discovery of HIV-1 integrase inhibitors by pharmacophore searching.

Abstract: Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152 compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206 876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 μM and are promising lead compounds for further HIV-1 integrase inhibitor development.

Hydrazide-Containing Inhibitors of HIV-1 Integrase†

Abstract: Inhibitors of HIV integrase are currently being sought as potential new therapeutics for the treatment of AIDS. A large number of inhibitors discovered to date contain the o-bis-hydroxy catechol structure. In an effort to discover structural leads for the development of new HIV integrase inhibitors which do not rely on this potentially cytotoxic catechol substructure, NSC 310217 was identified using a three-point pharmacophore search based on its assigned structure N-(2-hydroxybenzoyl)-N-(2-hydroxy-3-phenoxypropyl)hydrazine (1). When a sample of NSC 310217 was obtained from the NCI repository, it was shown to exhibit potent inhibition of HIV-1 integrase (3‘-processing IC50 = 0.6 μg/mL). In work reported herein, we demonstrate that NSC 310217, rather than containing 1, which has no inhibitory potency against HIV-1 integrase, is comprised of roughly a 1:1 mixture of N-(2-hydroxybenzoyl)-N‘-(2-hydroxy-3-phenoxypropyl)hydrazine (6) and N,N‘-bis-salicylhydrazine 7, with all inhibitory potency residing with com...

Arylamide inhibitors of HIV-1 integrase

Abstract: Based on data derived from a large number of HIV-1 integrase inhibitors, similar structural features can be observed, which consist of two aryl units separated by a central linker. For many inhibitors fitting this pattern, at least one aryl ring also requires ortho bis-hydroxylation for significant inhibitory potency. The ability of such catechol species to undergo in situ oxidation to reactive quinones presents one potential limitation to their utility. In an effort to address this problem, a series of inhibitors were prepared which did not contain ortho bis-hydroxyls. None of these analogues exhibited significant inhibition. Therefore an alternate approach was taken, whose aim was to increase potency rather than eliminate catechol substructures. In this latter study, naphthyl nuclei were utilized as aryl components, since a previous report had indicated that fused bicyclic rings may afford higher affinity relative to monocyclic phenyl-based systems. In preliminary work with monomeric units, it was found...

Potent Inhibitors of Human Immunodeficiency Virus Type 1 Integrase: Identification of a Novel Four-Point Pharmacophore and Tetracyclines as Novel Inhibitors

Abstract: A four-point pharmacophore was constructed from energy-minimized structures of chicoric acid and dicaffeoylquinic acid. The search of 206,876 structures in the National Cancer Institute 3D database yielded 179 compounds that contain this pharmacophore. Thirty-nine of these compounds were tested in an in vitro assay specific for human immunodeficiency virus type 1 integrase (IN). Each retrieved structure was fit to the pharmacophore, and the conformation that afforded the best fit was identified. Twenty of the 39 compounds tested exhibited IC50 values of < 20 microM. Among the most potent inhibitors, tetracyclines emerged as a new class of inhibitors. Although the parent tetracycline exhibited marginal potency against purified IN, all substituted tetracyclines tested showed 5-100-fold increased potency. Disintegration assays with truncated IN mutants indicated that tetracyclines inhibit the IN catalytic core domain. To investigate whether chelation of divalent metals is implicated in differential potency of tetracyclines, enzyme assays were performed in the presence of both Mn2+ or Mg2+; no significance difference in potency was observed. Rolitetracycline inhibited IN/DNA complex formation in the presence of EDTA, which suggests that inhibition was metal independent. Rolitetracycline reversed DNA binding of IN after the complex was allowed to form before the addition of drug. Selectivity of tetracyclines was also examined in an assay specific for topoisomerase I, and none of the tetracyclines tested induced topoisomerase I-mediated cleavable complex or inhibited camptothecin-induced cleavable complex. Remarkable potency against the IN in the absence of divalent metals and the core enzyme coupled with water solubility makes tetracyclines potential candidates for X-ray crystal structure determination with IN.

Mammalian DNA Topoisomerase I Mediates the Enhancement of Radiation Cytotoxicity by Camptothecin Derivatives

Abstract: The role of DNA topoisomerase I as a biochemical mediator of radiosensitization in cultured mammalian cells by camptothecin derivatives was studied. We found that, in Chinese hamster DC3F cells, camptothecin enhanced the cytotoxicity of radiation in a schedule-dependent manner. At 4 microM, a sensitizer enhancement ratio of 1.45 was observed when radiation was used concurrently with or immediately after drug treatment. By comparison, no enhancement was obtained if radiation preceded camptothecin treatment. Consistently, in human breast cancer MCF-7 cells, sensitizer enhancement ratios of 1.43, 1.38, and 1.05 were observed when radiation was used concurrently with, immediately after, or prior to treatment with 20(S)-10,11-methylenedioxycamptothecin (MDCamp). Three studies indicated that an intact stereospecific interaction between camptothecin derivatives and DNA topoisomerase I is essential in the induction of radiosensitization: (a) higher concentrations of camptothecin were required to radiosensitize the camptothecin-resistant DC3F/C-10 cells; (b) a newly identified topoisomerase 1-targeting Hoechst 33342 also radiosensitized DC3F cells; and (c) 20(S)-methylenedioxycamptothecin, but not its noncytotoxic 20(R)-stereoisomer, radiosensitized MCF-7 cells by obliterating the "shoulder" of the radiation survival curve. The mechanism of radiosensitization was investigated in DC3F cells. We found that camptothecin only minimally enhanced the cytoxic effect of radiation in G1-phase cells obtained by a mitotic shake-off technique as well as in plateau-phase cells arrested by growing to confluency. Our data suggest a potential development of topoisomerase I drugs as radiosensitizers in treating human malignancies.

HIV-1 Integrase as a Target for Antiviral Drugs:

Abstract: Retrovirus integration requires at least two viral components, one of the three retroviral enzymes, integrase, and cis-acting sequences at the ends of the retroviral DNA termini U3 and U5 ends of the long terminal repeats. Because the virus cannot replicate without integration into a host chromosome, integrase is a logical therapeutic target. Therapeutic inhibitors already exist for reverse transcriptase and protease, and attacking the virus on these sites together has already proven effective for combination therapy. Thus, the discovery of integrase inhibitors should provide an additional benefit. Screening and pharmacology of anti-integrase drugs is facilitated by the cloning and expression of recombinant retroviral integrases and their use in a series of in vitro assays that mimic integration in vivo. This review first describes the integration reactions in the retrovirus life cycle and the integrase protein. Then we provide a comprehensive review of the inhibitors identified to date.

Inactivation of p53 increases the cytotoxicity of camptothecin in human colon HCT116 and breast MCF-7 cancer cells.

Abstract: Camptothecin (CPT) derivatives are topoisomerase I (top1) inhibitors recently introduced as clinical agents. To explore the role of p53 in CPT-induced cytotoxicity, we examined CPT effects in two isogenic pairs of human cancer cell lines, MCF-7 breast carcinoma and HCT116 colon carcinoma cells, in which p53 function had been disrupted by transfection with the human papillomavirus type-16 E6 gene. Clonogenic survival assays showed that both MCF-7/E6 and HCT116/E6 cells were more sensitive to CPT. No differences in top1 protein levels and activity analyzed by a novel in vitro oligonucleotide assay were observed in the E6 transfectants. Also, CPT showed comparable top1 cleavable complex formation in vivo, as determined by DNA single-strand breaks and DNA protein cross-links. These results suggest that p53 can protect against CPT-induced cytotoxicity and that this protection is mediated downstream of CPT-induced DNA damage. Flow cytometry analyses showed that CPT can induce G1 arrest in cells with normal p53. This G1 arrest was markedly reduced in the p53-deficient cells. These results demonstrate a critical role of p53 as a G1 checkpoint regulator after CPT-induced DNA damage and suggest a rationale for the selectivity of CPT toward tumors with p53 mutations.

Differential cytotoxicity of clinically important camptothecin derivatives in P-glycoprotein-overexpressing cell lines.

Abstract: Camptothecin and its derivatives are specific inhibitors of eukaryotic topoisomerase I (top1) and are active in cancer patients against a variety of refractory solid tumors and leukemia. Purpose: The present study further investigated the relationship between multidrug resistance (MDR) mediated by P-glycoproteinMDR and potential resistance to camptothecin derivatives using two experimental systems: (1) MDR KB-V1 cells selected for vinblastine resistance, and (2) NIH3T3 cells transfected with a plasmid expressing wildtype P-glycoproteinMDR multidrug transporter (NIH-MDR-G185). Results: We found that both KBV-1 and NIH-MDR-G185 cells were resistant to topotecan, and that topotecan-induced cleavable complexes were reduced in KB-V1 cells, consistent with a role of P-glycoproteinMDR in cellular resistance to topotecan. By contrast, no significant resistance to camptothecin, 9-aminocamptothecin, 10, 11-methylenedioxycamptothecin, or SN-38 (the active metabolite of CPT-11) was observed in NIH-MDR-G185 cells, while KB-V1 cells were cross-resistant to these compounds but produced cleavable complexes similar to those produced by parental KB-3-1 cells. Conclusions: These results suggest that topotecan is the only camptothecin tested with significant susceptibility to MDR in cell culture, and that multidrug resistant cells such as KBV1 probably exhibit additional resistance mechanisms to camptothecins besides P-glycoproteinMDR overexpression.

Aclacinomycin A Stabilizes Topoisomerase I Covalent Complexes

Abstract: Aclacinomycin A (aclarubicin) is an anthracycline anticancer agent with demonstrated activity against both leukemias and solid tumors. Previous results suggested that a major activity of aclacinomycin A is the inhibition of topoisomerase II catalytic activity. We have applied a yeast system to test whether aclacinomycin A is a topoisomerase II inhibitor in vivo and to test whether we could identify other important targets of this drug. We have found that overexpression of yeast topoisomerase II confers resistance to aclacinomycin A in yeast, consistent with the hypothesis that this drug is a catalytic inhibitor of topoisomerase II. Interestingly, we have also found that in yeast, aclacinomycin A, like camptothecin, stabilizes topoisomerase I cleavage. We carried out biochemical analysis with purified human topoisomerase I and demonstrated that this drug efficiently stabilizes topoisomerase I covalent complexes, indicating that aclacinomycin A represents a novel class of combined topoisomerase I/II inhibitor.

DNA Topoisomerase II Inhibitors

Abstract: DNA topoisomerases represent a major focus of research not only for cancer chemotherapy, but also for gene regulation, cell cycle, mitosis, and chromosome structure. A number of reviews have been written on the subject (1–8).

Probing interactions between viral DNA and human immunodeficiency virus type 1 integrase using dinucleotides

Abstract: Retroviral integrases are essential for viral replication and represent an attractive chemotherapeutic target. In the current study, we demonstrated the activity of micromolar concentrations of dinucleotides against human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), simian immunodeficiency virus, and feline immunodeficiency virus integrases. The structure-activity relationship indicates that 5'-phosphorylation enhances potency and that phosphodiester and sugar modifications affect the inhibition of HIV-1 integrase. Base sequence selectivity was observed: pAC, pAT, and pCT were the most potent inhibitors, whereas pAA, pGA, and pGC showed low activity at 100 microM. The inhibition by pAC is consistent with the interaction of the enzyme with the 5' end of the noncleaved strand (5'-AC-3'). The linear and cyclic dinucleotides released by the 3'-processing reaction did not affect enzymatic activity at physiological concentrations. An increase in the length to trinucleotides or tetranucleotides enhanced potency by only 2-3-fold, suggesting that two neighboring bases may be sufficient for significant interactions. Inhibition of a truncated (50-212) integrase mutant and global inhibition of all nucleophiles in the 3'-processing reaction suggest that dinucleotides bind in the catalytic core. All of the active dinucleotides inhibited enzyme/DNA binding in their respective IC50 range. Although the dinucleotides tested showed no antiviral activity, these observations demonstrate the usefulness of dinucleotides in elucidating enzyme mechanisms and as potential ligands for cocrystallization and as lead structures for development of antivirals.

Novel 7-Alkyl Methylenedioxy-Camptothecin Derivatives Exhibit Increased Cytotoxicity and Induce Persistent Cleavable Complexes Both with Purified Mammalian Topoisomerase I and in Human Colon Carcinoma SW620 Cells

Abstract: An alkylating camptothecin (CPT) derivative, 7-chloromethyl-10,11-methylenedioxy-camptothecin (7-CM-MDO-CPT) was recently shown to produce irreversible topoisomerase I (top1) cleavage complexes by binding to the +1 base of the scissile strand of a top1 cleavage site. We demonstrate that 7-CM-EDO-CPT (7-chloromethyl-10,11-ethylenedioxy-camptothecin) also induces irreversible top1-DNA complexes. 7-CM-MDO-CPT, 7-CM-EDO-CPT, and the nonalkylating derivative 7-ethyl-10,11-methylenedioxy-camptothecin (7-E-MDO-CPT) also induced reversible top1 cleavable complexes, which were markedly more stable to salt-induced reversal than those induced by 7-ethyl-10-hyroxy-CPT, the active metabolite of CPT-11. This greater stability of the top1 cleavable complexes was contributed by the 7-alkyl and the 10,11-methylene- (or ethylene-) dioxy substitutions. Studies in SW620 cells showed that 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT are more potent inducers of cleavable complexes and more cytotoxic than CPT. The reversal of the cleavable complexes induced by 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT was markedly slower after drug removal than that for CPT, which is consistent with the data with purified top1. By contrast to CPT, 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT were cytotoxic irrespective of the presence of 10 μm aphidicolin. These results suggest that 7-E-MDO-CPT, 7-CM-MDO-CPT, and 7-CM-EDO-CPT are more potent top1 poisons than CPT and produce long lasting top1 cleavable complexes and greater cytotoxicity than CPT in cells.

2-Mercaptobenzenesulphonamides as novel inhibitors of human Immunodeficiency virus type 1 integrase and replication

Abstract: An obligatory requirement in the retroviral life cycle is the integration of the viral dsDNA into the host chromosome, a process performed by viral integrase. The retroviral integrase is able to catalyse at least three discrete enzymatic steps. Two of these steps, 3' processing and DNA strand transfer, can be measured in an in vitro assay in the presence of a duplex oligonucleotide corresponding to the viral long terminal repeat, recombinant integrase and the divalent cations, Mg 2+ or Mn 2+ . This assay provides an efficient means of testing integrase inhibitors. As part of our continuous effort in developing novel inhibitors we examined a series of 2-mercapto-benzenesulphonamides (MBSAs) for their inhibitory activity against human immunodeficiency virus type 1 (HIV-1) integrase. From the list of compounds tested in an assay specific for HIV-1 integrase, 26 compounds inhibited the 3' processing and strand transfer step with 50% inhibitory concentration (IC 50 ) values below 25 μM. All the thioether derivatives were inactive. These results were further compared with the ability of MBSAs to protect HIV-1-injected T4 lymphocyte CEM cells. Among 68 compounds tested, 27 exhibited antiviral activity in cell-based assays with therapeutic indices of 1-16. All the MBSAs with antiviral activity were also effective inhibitors of recombinant HIV-1 integrase. Several aromatic disulphides were also tested and found to exhibit moderate antiviral and anti-integrase activities. These data demonstrate that MBSAs can be developed as inhibitors of HIV-1 integrase with the potential for antiviral activity.

In vivo sequencing of camptothecin-induced topoisomerase I cleavage sites in human colon carcinoma cells

Abstract: Camptothecin (CPT) is a specific topoisomerase I (top1) poison which traps top1 cleavable complexes; e.g. top1-linked DNA single-strand breaks with 5'-hydroxyl and 3'-top1 linked termini. CPT is also a potent anticancer agent and several of its derivatives have recently shown activity in the chemotherapy of solid tumors. Our aim was to apply the ligation-mediated polymerase chain reaction (LM-PCR) method to DNA extracted from CPT-treated cells in order to: (i) evaluate LM-PCR as a sensitive technique to detect in vivo CPT-induced cleavable complexes; (ii) investigate the frequency and distribution of CPT-induced DNA damage in vivo ; and (iii) compare the distribution and intensity of cleavage sites in vivo and in vitro. This report describes a protocol allowing the sequencing of top1-mediated DNA strand breaks induced by CPT in the coding strand of the 18S rRNA gene of human colon carcinoma cells. CPT or its clinical derivatives, topotecan, CPT-11, SN-38, and 9-aminocamptothecin differed in their potency and exhibited differences in their DNA cleavage pattern, which is consistent with our previous in vitro studies [Tanizawa et al . (1995) Biochemistry , 43, 7200-7206]. CPT-induced DNA cleavages induced in the presence of purified top1 were induced at the same sites in the human 18S rDNA. However, the relative intensity of the cleavages were different in vivo and in vitro. Because mammalian cells contain approximately 300 copies of the rDNA gene per genome, rDNA could be used to monitor CPT-induced DNA cleavage in different cell lines and possibly in tumor samples.

Sers and Fluorescence Study of the Molecular Interactions of Camptothecins with DNA and DNA Topoisomerase I and in their Ternary Cleavable Complexes

Abstract: Potent antitumour effect of camptothecins (CPTs) has been shown to correlate with trapping of the nuclear enzyme, DNA topoisomerase I (top 1), in the drug/top1/DNA cleavable complexes [1, 2]. CPTs are selectively active at top1 cleavage sites bearing a guanine residue at the 5’-terminus of the enzyme-mediated DNA break [3]. Up to now, there are no published data on the molecular mechanisms of the cleavable complexes formation by camptothecins. Structure-activity studies indicate that one of the important structural requirements for CPTs efficacy is the hydrolysable α-hydroxy-lactone ring of these drugs [2]. Hydrolysed open-ring species (carboxylate forms) of CPTs are inactive and toxic [2]. To study both the qualitative and quantitative aspects of CPT-DNA, CPT-top1, CPT-top1-DNA molecular interactions, we have applied the spectroscopic approach that combines surface-enhanced Raman scattering (SERS) and fluorescence emission techniques