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Stephen Neidle

Publications and source records attributed to Stephen Neidle.

50 records · Page 3Linked to original sources
PubMed2003

The structure of telomeric DNA.

The telomere is a nucleoprotein complex located at the ends of eukaryotic chromosomes. It is essential for maintaining the integrity of the genome. It is not a linear structure and, for much of the cell cycle, telomeric DNA is maintained in a loop structure, which serves to protect the vulnerable ends of chromosomes. Many of the key proteins in the telomere have been identified, although their interplay is still imperfectly understood and structural data are only available on a few. Telomeric DNA itself comprises simple guanine-rich repeats for most of its length, culminating in a short overhang of single-stranded sequence at the extreme 3' ends. This can, at least in vitro, fold into a wide variety of four-stranded quadruplex structures, many of whose arrangements are being revealed by crystallographic and NMR studies.

DNA↗
PubMed2003

DNA sequence specificity of triplex-binding ligands.

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n. In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)n (CCT)n and (TTC)n. 2,7-Anthraquinone stabilizes (TC)n (CCT)n and (TTC)n, although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)n are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)n and neither affects the complex with (CT)n. We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C+GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C+GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.

Anthraquinones↗
PubMed2003

Acquired cellular resistance to flavopiridol in a human colon carcinoma cell line involves up-regulation of the telomerase catalytic subunit and telomere elongation. Sensitivity of resistant cells to combination treatment with a telomerase inhibitor.

Flavopiridol is a broad-spectrum inhibitor of cyclin-dependent kinases and of global transcription via the inhibition of positive transcription elongation factor b (P-TEFb). Although flavopiridol is currently undergoing phase II clinical trials, acquired cellular resistance to the compound during treatment is a potential problem, as it is with almost all current anticancer agents. A HCT116 human colon carcinoma cell line with an acquired 8-fold resistance to flavopiridol has been established. We report here that there are changes in these resistant cells in terms of telomere length and telomerase activity, whereas no change in the expression of the P-TEFb subunits CDK9, cyclin T1, cyclin T2a, or cyclin T2b was observed. The level of mRNA expression for the telomerase catalytic subunit hTERT was increased over 2-fold in the resistant cells, and mean telomere length was found to be 2 kb longer than the parental length, although telomerase activity was unchanged. The level of mRNA expression for the telomeric binding protein Pot1 was also increased. We also report that treatment of HCT116 cells with a combination of the G-quadruplex interacting telomerase inhibitor BRACO-19 and flavopiridol results in a 3-fold decrease in population doubling and prevents recovery from treatment with either compound alone. Treatment of flavopiridol-resistant cells with BRACO-19 alone also led to rapid inhibition of cell growth, which is not observed in the parental line. The finding that only the resistant line, with up-regulated telomerase, responds to this G-quadruplex inhibitor is consistent with the hypothesis that the mechanism of BRACO-19 down-regulation of cell growth directly involves the targeting of telomeres and telomerase.

Acridines↗
PubMed2003

The design of G-quadruplex ligands as telomerase inhibitors.

Guanine-rich repetitive DNA sequences are of particular importance at the ends of chromosomes, where they are associated with a number of proteins to form telomeres. Their function is in large part to protect chromosomal ends from unwanted degradation and chromosomal fusions, although in normal somatic cells telomeres progressively shorten, eventually becoming non-proliferating and consequently these cells have a finite lifetime. By contrast tumour cell telomeres are maintained in length so that tumour cells are effectively immortalised. The reverse transcriptase enzyme telomerase is activated in over 80% of tumour cells, and it undertakes the synthesis of further telomeric DNA repeats, so directly maintaining telomeres. The inhibition of telomerase leads to the senescence and eventual apoptosis of tumour cells, and thus telomerase is an attractive target for selective chemotherapy. This review describes an approach to the inhibition of telomerase that involves the folding of telomeric DNA into a four-stranded quadruplex structure, held together by Hoogsteen hydrogen-bonded arrays of guanine bases. The formation of a quadruplex structure at the 3' end of telomeric DNA effectively hinders telomerase from adding further repeats. A number of small-molecule ligands are described that stabilise quadruplex formation, and which result in telomerase inhibition. Implications for anti-tumour therapy with such molecules are discussed, and the particular challenges and problems discussed.

Computer Simulation↗
PubMed2003

Telomerase inhibitors in cancer therapy: current status and future directions.

The ends of chromosomes (telomeres) are subject to progressive shortening in normal somatic cells, leading ultimately to irreversible growth arrest. In contrast, telomeres in all cancer cells are stabilized in length and effectively immortalized by the enzyme telomerase, which catalyzes the synthesis of telomeric DNA repeats. Several strategies have been devised for the inhibition of telomerase in the hope that this will result in anticancer effects. The principal approaches of catalytic inhibitors, antisense to the template, and folding of the DNA substrate, are reviewed and critically evaluated for their potential in anticancer therapy.

Animals↗
PubMed2002

Strong binding in the DNA minor groove by an aromatic diamidine with a shape that does not match the curvature of the groove.

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.

DNA↗
PubMed2002

Solid-phase synthesis of symmetrical 3,6-bispeptide-acridone conjugates.

[reaction: see text] A novel high-yielding method for the solid-phase synthesis of 3,6-bispeptide-acridone conjugates is reported. It involves initial coupling of bifunctionalized acridone to a resin-bound peptide followed by an on-bead site-site reaction to couple the second peptide. This method leads to clean symmetrical bispeptide derivatives and appears to be general. This strategy will enable the generation of a library of 3,6-bispeptide-acridones to be screened for selective binding to telomeric G-quadruplex DNA.

Acridines↗
PubMed2002

Crystal structure of the potassium form of an Oxytricha nova G-quadruplex.

The crystal structures of the potassium-containing quadruplex formed from the Oxytricha nova sequence d(GGGGTTTTGGGG) are reported, in two space groups, the orthorhombic P2(1)2(1)2(1) and the trigonal P3(2)21, which diffract to 2.0 A and 1.49 A, respectively. The orthorhombic form contains two independent quadruplexes in the asymmetric unit, and the trigonal form contains one. All three of these quadruplexes adopt an identical fold, with two strands forming an antiparallel diagonal arrangement. This is identical with that observed previously in NMR studies of the native sodium and potassium forms, and a crystallographic analysis of it complexed with an O. nova protein. The present analysis demonstrates that the native structure is the same in solution and in the crystalline state and, moreover, that the nature of the counter-ion does not affect the overall fold of this quadruplex. The analysis corrects an earlier crystallographic study of this quadruplex. The conformation of the tetra-thymine loop is described in detail, which involves the third thymine base folding back to interact with the first thymine base. The water networks in the grooves and loops are described and, in particular, the ability of water molecules to form a continuous spine of hydration in the narrow groove is detailed. Each quadruplex has five potassium ions organised in a linear channel, with square antiprismatic coordination to each ion from oxygen atoms.

Animals↗
PubMed2002

Crystal structure of parallel quadruplexes from human telomeric DNA.

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, are fundamental in protecting the cell from recombination and degradation. Disruption of telomere maintenance leads to eventual cell death, which can be exploited for therapeutic intervention in cancer. Telomeric DNA sequences can form four-stranded (quadruplex) structures, which may be involved in the structure of telomere ends. Here we describe the crystal structure of a quadruplex formed from four consecutive human telomeric DNA repeats and grown at a K(+) concentration that approximates its intracellular concentration. K(+) ions are observed in the structure. The folding and appearance of the DNA in this intramolecular quadruplex is fundamentally different from the published Na(+)-containing quadruplex structures. All four DNA strands are parallel, with the three linking trinucleotide loops positioned on the exterior of the quadruplex core, in a propeller-like arrangement. The adenine in each TTA linking trinucleotide loop is swung back so that it intercalates between the two thymines. This DNA structure suggests a straightforward path for telomere folding and unfolding, as well as ways in which it can recognize telomere-associated proteins.

Base Sequence↗
PubMed2002

Protein and drug interactions in the minor groove of DNA.

Interactions between proteins, drugs, water and B-DNA minor groove have been analyzed in crystal structures of 60 protein-DNA and 14 drug-DNA complexes. It was found that only purine N3, pyrimidine O2, guanine N2 and deoxyribose O4' are involved in the interactions, and that contacts to N3 and O2 are most frequent and more polar than contacts to O4'. Many protein contacts are mediated by water, possibly to increase the DNA effective surface. Fewer water-mediated contacts are observed in drug complexes. The distributions of ligands around N3 are significantly more compact than around O2, and distributions of water molecules are the most compact. Distributions around O4' are more diffuse than for the base atoms but most distributions still have just one binding site. Ligands bind to N3 and O2 atoms in analogous positions, and simultaneous binding to N3 and N2 in guanines is extremely rare. Contacts with two consecutive nucleotides are much more frequent than base-sugar contacts within one nucleotide. The probable reason for this is the large energy of deformation of hydrogen bonds for the one nucleotide motif. Contacts of Arg, the most frequent amino acid ligand, are stereochemically indistinguishable from the binding of the remaining amino acids except asparagine (Asn) and phenylalanine (Phe). Asn and Phe bind in distinct ways, mostly to a deformed DNA, as in the complexes of TATA-box binding proteins. DNA deformation concentrates on dinucleotide regions with a distinct deformation of the delta and epsilon backbone torsion angles for the Asn and delta, epsilon, zeta and chi for the Phe-contacted regions.

Amino Acids↗
PubMed2002

Telomere maintenance as a target for anticancer drug discovery.

Maintenance of telomeres--specialized complexes that protect the ends of chromosomes--is undertaken by the enzyme complex telomerase, which is a key factor that is activated in more than 80% of cancer cells that have been examined so far, but is absent in most normal cells. So, targeting telomere-maintenance mechanisms could potentially half tumour growth across a broad spectrum of tumour types, with little cytotoxic effect outside tumours. Here, we describe the current understanding of telomere biology, and the application of this knowledge to the development of anticancer drugs.

Animals↗
PubMed2002

A G-quadruplex-interactive potent small-molecule inhibitor of telomerase exhibiting in vitro and in vivo antitumor activity.

The telomerase complex is responsible for telomere maintenance and represents a promising cancer therapeutic target. We describe herein the antitelomerase and antitumor properties of a small-molecule compound designed by computer modeling to interact with and stabilize human G-quadruplex DNA, a structure that may form with telomeric DNA, thereby inhibiting access to telomerase. The 3,6,9-trisubstituted acridine 9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis(3-pyrrolodinopropionamido) acridine (BRACO19) represents one of the most potent cell-free inhibitors of human telomerase yet described (50% inhibitory concentration of 115 +/- 18 nM). Moreover, in contrast to G-quadruplex interactive agents described previously, BRACO19 did not cause nonspecific acute cytotoxicity at similar concentrations to those required to completely inhibit telomerase activity. There exists a 90-fold differential (mean 50% inhibitory concentration for acute cell kill across seven human tumor cell lines of 10.6 +/- 0.7 microM). The exposure of 21NT human breast cancer cells, which possess relatively short telomeres, to nonacute cytotoxic concentrations of BRACO19 (2 microM) resulted in a marked reduction in cell growth after only 15 days. This was concomitant with a reduction in intracellular telomerase activity and onset of senescence as indicated by an increase in the number of beta-galactosidase positive-staining cells. Intraperitoneal administration of nontoxic doses of BRACO19 (2 mg/kg) to mice bearing advanced stage A431 human vulval carcinoma subcutaneous xenografts and previously treated with paclitaxel induced a significant increase in antitumor effect compared with that observed with paclitaxel alone. BRACO19 thus represents the first of a "second generation" of G-quadruplex-mediated telomerase/telomere-interactive compounds. It possesses nanomolar potency against telomerase but low nonspecific cytotoxicity, growth inhibitory effects, and induction of senescence in a human breast cancer cell line and, moreover, significant antitumor activity in vivo when administered post paclitaxel to mice bearing a human tumor xenograft carcinoma.

Acridines↗
PubMed1996

Synthesis of Sequence-Selective C8-Linked Pyrrolo[2,1-c][1,4]benzodiazepine DNA Interstrand Cross-Linking Agents.

An efficient convergent synthesis of a homologous series of C8-linked pyrrolobenzodiazepine dimers with remarkable DNA interstrand cross-linking activity and potent in vitro cytotoxicity is reported. The "amino thioacetal" cyclization procedure was used to produce the electrophilic DNA-interactive N10-C11 imine moiety during the final synthetic step. In order to construct the key A-ring fragments (9a-d), a versatile convergent approach has been developed to join two units of vanillic acid with alpha,omega-dihaloalkanes of varying length to provide the required bis(4-carboxy-2-methoxyphenoxy)alkanes while avoiding the formation of mixtures of monoalkylated and bisalkylated products.

Journal Article↗