Biomedical subjects
Publications and source records attributed to G Wickham.
The solid phase synthesis of a set of peptide aldehydes derived from the NS5A/NS5B junction of hepatitis C virus (HCV) viral polyprotein is demonstrated using an oxazolidine linker and the Multipin method. Deletion of the P6 and P5 residues results in a dramatic loss of inhibitory activity.
Solid-phase synthesis is greatly dependent on the solid phase. We are interested in the development of a "pellicular" type of solid support where a more mobile polymer is grafted to rigid plastics. Compared to low cross-linked microporous beads that dominate the field, this approach allows great flexibility of design, as plastics are available as sheets, films, or threads, or can be molded into any shape, as required. Many different polymers or copolymers can be grafted onto any particular shape to give a wide choice of options in the physicochemical characteristics of the actual solid support. As an example of such a solid support, we report on polystyrene-grafted polypropylene in a particular shape that we have called "Lanterns." Its synthesis characteristics are compared to the commonly available low cross-linked polystyrene resins. As well, the handling advantages of these types of supports in multiple synthesis are highlighted.
The utility of electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) for the characterisation of ligand-oligonucleotide adducts is demonstrated with adducts formed between the oligonucleotide 5'-CACGTG-3' and both a platinating agent, cis-diamminedichloroplatinum(II) (cisplatin), and an alkylating ligand, n-bromohexylphenanthridinium bromide (phenC6Br). We have demonstrated previously that negative ion MS/MS spectra of alkylated oligonucleotides show a highly specific fragmentation pathway that enables the site of binding of the ligand to be readily identified. In comparison, the positive ion ESI-MS/MS spectra reported here also show a single major fragmentation pathway, but the dominant ion is the protonated ligand-base adduct. MS/MS of this ion confirms the site on binding of the ligand to the guanine base. MS/MS spectra of cisplatin adducts show much less specific fragmentation than alkylated adducts, particularly in the negative ion mode. This suggests that the ESI-MS/MS spectra of ligand-DNA adducts are strongly influenced by the extent to which the ligand weakens the glycosidic bond in the residue to which it is bound. For platinating agents, which do not labilise the glycosidic bond, additional experiments involving MS/MS of source-generated product ions were required to enable isomeric adducts to be distinguished.
Cisplatin analogues were synthesised that consisted of platinum(II) diamine complexes tethered via a polymethylene chain ( n = 3, 5, 8 and 10) to a phenanthridinium cation. Both chloro and iodo leaving groups were examined. DNA adduct formation was quantitatively analysed using a linear amplification system with the plasmid pGEM-3Zf(+). This system utilised Taq DNA polymerase to extend from an oligonucleotide primer to the damage site. This damage site inhibited the extension of the DNA polymerase. The products were electrophoresed on a DNA sequencing gel enabling adduct formation to be determined at base pair resolution. The damage intensity at each site was determined by densitometry. The platinum phenanthridinium complexes were shown to damage DNA at shorter incubation times than cisplatin. To produce similar levels of damage, an 18 h incubation was required for cisplatin compared to 30 min for the n = 3 platinum phenanthridinium complexes; this indicates that the intercalating chromophore causes a large increase in the rate of platination. A reaction mechanism involving direct displacement of the chloride by the N-7 of guanine may account for the rate increase. These results indicate that further development of these compounds could lead to more effective cancer chemotherapeutic agents.
2D NMR spectroscopic methods have been used to determine the structure of the adduct formed between the antitumor antibiotic hedamycin and the oligodeoxyribonucleotide duplex d(CACGTG)2. Evidence for both intercalation and alkylation in the adduct was observed, and a model for the binding interaction was constructed based on intermolecular NOEs and distance-restrained molecular dynamics. In our computationally refined model, the anthrapyrantrione chromophore of hedamycin is intercalated between the 5'-CG-3' bases with the two aminosugar groups placed in the minor groove and the six carbon bisepoxide side chain located in the major groove. The anglosamine sugar attached at C8 is oriented in the 3' direction relative to the intercalation site, while the N,N-dimethylvancosamine attached at C10 is oriented to the 5' side, with each aminosugar wedged between a guanine exocyclic amino group and one of the groove walls. The terminal epoxide carbon C18 is covalently bound to the N7 atom of the central guanine, as evidenced by lability of the C8 hydrogen of this purine upon reaction with hedamycin. Our binding model places the C10-attached N,N-dimethylvancosamine of hedamycin in van der Waals contact with the alkylated strand. A strong NOE contact verifies the close proximity of the terminal methyl group (C19) of the bisepoxide side chain to the methyl group of the thymine on the 3' side of the alkylated guanine. This, in conjunction with other data, suggests hydrophobic interactions between the bisepoxide chain and the floor of the major groove may contribute to sequence recognition. Furthermore, it is proposed that the 5'-CGT sequence selectivity of hedamycin arises, in part, from complementarity in shape between the chromophore substituents and the major and minor groove at the binding site.
The sequence specificity of DNA damage of n-bromoalkylphenanthridinium bromides, with linker chain lengths (n) of 4,6,8 and 10 methylene groups, was investigated in the plasmid pUC8 and in intact human cells. A linear amplification assay was used to elucidate the DNA sequence specificity of the alkylating agents. In this assay Taq DNA polymerase extends from an oligonucleotide primer up to the damage site and the products run on a DNA sequencing gel to reveal the precise sites of DNA damage. For both the plasmid and cellular experiments, the compound that caused the most damage to DNA was the n=6 compound, followed by (in decreasing order) the n=4, n=8, and n=10 compounds. There were significant differences in the sequence specificity of DNA damage between n-bromoalkylphenanthridinium bromides of different linker chain length; (1) the main sites of damage were at guanines for the n=4,6 and 8 compounds but at guanines and adenines for the n=10 compound; (2) a consensus sequence of 5'-c(a/t)Ggg-3' was obtained for the n=4,6 and 8 compounds but 5'-c(a/c)(G/A)(g/a)-3' for the n=10 compound; (3) runs of consecutive Gs were the major site of damage for the n=4,6 and 8 compounds, but consecutive Gs or consecutive As for the n=10 compound; (4) for damage at single isolated guanines, the most damaged sequences were at 5'-Ga-3' for the n=4 compound but at 5'-Gt-3' for the n=6,8 and 10 compounds. The tandemly repeated alpha RI DNA sequence was the DNA target in intact human K562 cells. In intact human cells, the compounds produced damage with similar DNA sequence selectivity to that found in plasmid DNA. The n=4 and 6 compounds possess marginal anti-tumour activity and these compounds produced the most damage in intact human cells. The n=8 and 10 compounds do not demonstrate significant anti-tumour activity and these compounds resulted in the least damage in cells.
The sequence specificity of the pluramycin antibiotics hedamycin and DC92-B, was established in intact human cells using a linear amplification system. In this system an oligonucleotide primer is extended by Taq DNA polymerase up to a damage site. The products are run on a DNA sequencing gel and the damage can be determined to the exact base pair. The human repetitive alpha RI DNA was used as the target DNA sequence for these experiments. It was found that G residues were the main site of adduct formation, for both hedamycin and DC92-B. The sequences 5'-TGT and 5'-CGT were the most intense sites of DNA damage. A comparison of the DNA damage intensity in intact cells and purified DNA revealed that the sequence position of adduct formation was very similar in the two environments. However, a densitometric comparison of the damage intensity in the two environments revealed significant differences. Two regions were found (120 and 130 bp in length) where the damage intensity was relatively lower in intact cells compared to purified DNA. But at the boundaries of these sequences, there were regions (approx. 50-60 bp long) that were relatively more damaged in intact cells compared to purified DNA. One explanation of this phenomenon is the presence of a protecting nucleosome core on each of the 120/130 bp regions and flanking nucleosome linker regions of 50-60 bp. This postulated sequence phasing of the nucleosomes corresponds almost exactly with the major nucleosome phasing found in African green monkey cells. Also the centromere protein B binding site is found in the border region between the nucleosome core and linker DNA regions. Hedamycin and DC92-B produced nearly identical results in this human cell system.
The sequence selectivity of DNA alkylation by the recently isolated pluramycin antitumour antibiotic DC92-B has been investigated using two methods: a piperidine-induced strand-breaking procedure and a Taq DNA polymerase/linear amplification method. These techniques reveal that guanines are the most reactive sites for alkylation and that the level of adduct formation at these sites is clearly sequence dependent. The highest levels of alkylation occurred at isolated guanines located in 5'-CGT sequences and also at the 5'-G in some 5'-CGG sequences. Isolated guanines in 5'-TGT sequences were also quite reactive. We have also re-examined, in parallel, the sequence selectivity of binding of the structurally-related compound hedamycin: the first known example of a bis(epoxide)-containing, DNA-alkylating pluramycin. Our studies included a more extensive sequence analysis of hedamycin binding than that previously reported and we are able, therefore, to define more precisely the sequence preference. Despite significant differences in the stereochemistry and substitution of their bis(epoxide) sidechains, hedamycin and DC92-B exhibited very similar sequence selectivities in our assays.
Covalent binding of the antitumour antibiotic hedamycin to the self-complementary hexadeoxyribonucleotide 5'-CACGTG-3' has been investigated by electrospray ionization mass spectrometry (ESI-MS). Ions due to double-stranded forms of the free 5'-CACGTG-3' and the hedamycin-5'- CACGTG-3' adduct have been observed in ESI mass spectra and their identity has been confirmed by resolution of individual charge states in ESI-MS spectra. Clear evidence that specific base-paired associations are being observed in ESI-MS is provided by the results of a titration experiment involving alkylated and non-alkylated complementary strands. This work demonstrates the potential of this powerful new tool for studying ligand-DNA binding.
A series of terephthalamide derivatives with the substituents R = NO2, NH2 and NHCOCH2NH2 on the central aryl ring have been synthesized, and their interaction with the DNA decamer d(GGTAATTACC)2 has been studied by 1H NMR. The amine and nitro (R = NH2, NO2) derivatives bind with micromolar affinities and exhibit NMR spectra characteristic of fast exchange on the chemical shift time scale. The glycine derivative (R = NHCOCH2NH2) binds more tightly and a number of its resonances are in intermediate to slow exchange on the chemical shift time scale. Estimates of binding affinities and bound chemical shifts of ligand and DNA resonances were made from an analysis of chemical shifts and linewidths in a series of spectra with ligand duplex mole ratios ranging from 0:1 to 2:1. The data unequivocally suggest that all three ligands bind in the minor groove of the DNA decamer and more specifically that the binding site is localized over the ATTA sequence. The ligand is able to exchange rapidly between two symmetry-related ATTA sites per decamer.
Electrospray ionization mass spectrometry (ESI-MS) has been used to examine the covalent binding of the anti-tumour agents cisplatin and hedamycin with self-complementary oligonucleotides 5'-TACGTA-3', 5'-CACGTG-3', 5'-AGGCCT-3' or 5'-CGTACG-3' as models for binding to cellular DNA. The observation of duplex forms of oligonucleotide adducts of these compounds in the gas phase has been found to correlate with the stability of the adducts in solution. Hedamycin, which both intercalates into and alkylates DNA, enhances the stability of duplex ions in ESI mass spectra. In contrast, the binding of cisplatin is known to destabilise duplexes in solution and only weak double-stranded peaks are observed in the ESI spectra of cisplatin-oligonucleotide adducts. Results of titration experiments with the hedamycin-5'-CACGTG-3' adduct and complementary and non-complementary oligo-nucleotides provide strong evidence that the observed duplex ions are the result of specific base-paired associations in the gas phase, rather than non-specific interactions. Finally, estimates of the extent of cisplatin binding to different sequences based on ESI mass spectra of crude reaction mixtures are found to correlate well with data obtained by reversed-phase high-performance liquid chromatography. This work demonstrates the considerable potential of ESI-MS as a tool for characterization of the interactions of antitumour agents with DNA.
Bidirectional transcription footprinting has been used to probe the platination of DNA by cisplatin, and to examine the modulation of these interactions by (a) cyclisation of the non-reactive amino group by either ethyl or propyl groups, and (b) the further addition of a pendant intercalator (9-amino acridine) linked by either phenylethyl or phenylpentyl groups. Intrastrand crosslinking was detected for all derivatives at all 5'-GG and 5'-AG sequences on the template strand, but the same sites did not result in transcriptional blockages when on the non-template strand. There was little effect of cyclysation of the amino groups, but the further addition of an intercalator resulted in three responses: a time-dependent increase of the blocked transcript by one and three nucleotides; a reduction of the sequence selectivity of platination; a decrease of apparent interstrand crosslinking for these derivatives with a pendant intercalator tethered to the amino moiety of cisplatin.
The sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II) (cisplatin) and four analogues in human (HeLa) cells was studied using Taq DNA polymerase and a linear amplification system. The primer extension is inhibited by the drug-DNA adducts, and hence the sites of these lesions can be analyzed on DNA sequencing gels. The repetitive alphoid DNA was used as the target DNA in human cells. A comparison was made between adduct formation in human cells and in purified DNA. The sequence-specific position and relative intensity of damage was similar in both systems for cisplatin, dichloro(ethylenediammine)platinum(II) (PtenCl2), and N-[3-N-(ethylenediamino)propyl]acridine-4-carboxamidedichloropl atinum(II) (4AcC3PtenCl2). However, no DNA damage could be detected in cells for trans-diamminedichloroplatinum(II) (transPt) or N-[3-N-(ethylenediamino)propyl]acridine-2-carboxamide-dichloroplat inum(II) (2AcC3PtenCl2) despite the ability of these latter analogues to damage purified DNA. Cisplatin, PtenCl2, and 4AcC3PtenCl2, which significantly damaged DNA inside cells, also show antitumor activity in mouse models. However, transPt and 2AcC3PtenCl2, which did not detectably damage DNA inside cells, did not show such antitumor activity. This correlation between intracellular DNA damaging ability and in vivo antitumor activity indicates the potential use of the human cells/Taq DNA polymerase/linear amplification technique as a convenient method for screening new cisplatin analogues for useful chemotherapeutic activity.
cis-Diamminedichloroplatinum(II) (cisplatin) forms adducts with DNA. The sequence specificity of formation of cisplatin adducts with plasmid DNA was investigated using Taq DNA polymerase. This procedure involved the extension of an oligonucleotide primer by Taq DNA polymerase up to the cisplatin adduct. Using thermal cycling, this process is repeated many times in order to amplify the signal. The products of this linear amplification can then be examined on DNA sequencing gels, and the sequence specificity of cisplatin adduct formation can be determined to the exact base pair. In the pUC8 plasmid, the sequences that produced the most intense damage sites (as determined by densitometry) were runs of two or more Gs. Adducts could also be detected at GA, AG, and GC dinucleotides. Four other cisplatin analogues were also tested in the system. Two of these analogues contained an attached intercalating chromophore, and the strong damage with these compounds was similar to that found for cisplatin, but the medium and weak damage tended to be different. Weak damage was also detected with trans-diamminedichloroplatinum(II). With this compound, a large number of the damage sites were at the CG dinucleotide. This technique represents a simple, accurate, and quick method for determining the sequence specificity of damage for a cisplatin analogue in any DNA sequence.
A series of acridine-2- and -4-carboxamide-linked analogues of PtenCl2 has been prepared and evaluated for biological activity against several tumor cell lines in vitro and in vivo. The platinum complexes were generally more cytotoxic than the corresponding ligands against wild-type P388 leukemia cells in vitro, with acridine-4-carboxamide complexes being the more effective. In contrast to cisplatin and PtenCl2, the complexes were equally active in vitro against both wild-type and cisplatin-resistant P388 lines. The 4-carboxamide complexes showed high levels of in vivo activity (ILS greater than 100%) against wild-type P388 using a single-dose protocol, and one compound was also significantly active in vivo in a cisplatin-resistant line, against which cisplatin and PtenCl2 are inactive.
A series of dimers of the monofunctional platinum species [Pt(dien)Cl]+, linked by a variety of flexible (polymethylene) and more rigid chains, was prepared and evaluated for DNA interactions and cytotoxic activity. The polymethylene-linked dimers were prepared by acylation of N(1),N(3)-bistrityldiethylenetriamine with alpha, omega-dicarboxylic acid chlorides, followed by reduction with diborane. Platination of these ligands was achieved with K2PtI4 prepared in situ, followed by anion exchange. Solutions of the bis(Pt(dien)Cl)2+ complexes were stable, and shown to be pure by 195Pt NMR, but solid products could not be isolated. All of the bis(Pt(dien)Cl)2+ complexes unwound closed circular supercoiled DNA more efficiently than the monomer, and were more efficient than the difunctional platinum complex cisplatin at cross-linking linearized plasmid DNA, as measured on non-denaturing agarose gels. None of the bis(Pt(dien)Cl)2+ complexes were as cytotoxic as cisplatin in both the wild-type and platinum-resistant P388 murine leukaemia cell lines. The more rigid analogues were equitoxic in both sensitive and cisplatin-resistant cells, but none showed in vitro activity against the P388 tumour.
We have synthesised an homologous series of n-bromoalkylphenanthridinium bromides and studied their DNA-binding and antitumour properties. Each of these compounds has the capacity both to intercalate and alkylate DNA. Dialysis measurements reveal a relatively high affinity for calf thymus DNA, being about 10(5) M-1 at ionic strength 0.01. Incubating calf thymus DNA-ligand complexes having a ligand-to-basepair ratio of 0.4 at 37 degrees C for 18 h leads to maximum alkylation levels of about one ligand molecule bound irreversibly per 40 basepairs. The reactivity of these compounds towards DNA is chain-length dependent, the n-decyl compound, for example, requiring about 10-times the ligand-to-basepair input ratio of the n-hexyl derivative to reach the same level of alkylation. The limited degree of alkylation is a consequence of conversion of the alkylbromides to the less reactive alkylchlorides in the buffer medium. The results of DNA sequencing experiments indicate that the n-hexyl derivative alkylates at guanines occurring in 5'-GT-3' sequences and in runs of guanines [(Gp)n]. The corresponding n-decyl compound, on the other hand, is highly selective for guanines in 5'-GT-3' sequences only and also reacts weakly with some adenines. None of the phenanthridinium compounds showed significant antitumour activity in the P388 murine leukaemia test system.
The interaction of a new group of 'quinomycin-like' antibiotics with the DNA duplexes d(ACGT)2 and d(GACGTC)2 has been investigated in solution by 1H NMR spectroscopy. By monitoring the intensity of intranucleotide base H6/H8 to deoxyribose H1'NOE cross-peaks we conclude that the terminal A-T basepairs flanking the CG bisintercalation site in the d(ACGT)2 complex adopt the Hoogsteen bonding scheme, with the purine base in a syn conformation. By comparison in the d(GACGTC)2 complex all glycosidic bond angles are anti, consistent with a preferred Watson-Crick basepairing scheme. Both DNA duplexes appear to be significantly unwound compared with the ligand-free DNAs. The data illustrate the influence of helical constraints on the stability of the Hoogsteen bonding scheme adjacent to the drug binding sites.