An extended pyrrolobenzodiazepine-polyamide conjugate with selectivity for a DNA sequence containing the ICB2 transcription factor binding site.

The binding of nuclear factor Y (NF-Y) to inverted CCAAT boxes (ICBs) within the promoter region of DNA topoisomerase IIα results in control of cell differentiation and cell cycle progression. Thus, NF-Y inhibitory small molecules could be employed to inhibit the replication of cancer cells. A library of pyrrolobenzodiazepine (PBD) C8-conjugates consisting of one PBD unit attached to tri-heterocyclic polyamide fragments was designed and synthesized. The DNA-binding affinity and sequence selectivity of each compound were evaluated in DNA thermal denaturation and DNase I footprinting assays, and the ability to inhibit binding of NF-Y to ICB1 and ICB2 was studied using an electrophoretic mobility shift assay (EMSA). 3a was found to be a potent inhibitor of NF-Y binding, exhibiting a 10-fold selectivity for an ICB2 site compared to an ICB1-containing sequence, and showing low nanomolar cytotoxicity toward human tumor cell lines. Molecular modeling and computational studies have provided details of the covalent attachment process that leads to formation of the PBD-DNA adduct, and have allowed the preference of 3a for ICB2 to be rationalized.

Inhibition of DNA binding of the NF-Y transcription factor by the pyrrolobenzodiazepine-polyamide conjugate GWL-78.

Many genes involved in cell cycle control have promoters that bind the heterotrimeric transcription factor NF-Y. Several minor-groove binding drugs have been shown to block interactions of transcription factors with cognate DNA-binding sequences. We showed previously that noncovalent minor-groove binding agents block interactions of NF-Y with the promoter of topoisomerase IIalpha (topo IIalpha). In this study, we investigated the ability of GWL-78, a pyrrolobenzodiazepine-poly(N-methylpyrrole) conjugate, to inhibit the binding of NF-Y to DNA. Electrophoretic mobility shift assays showed that GWL-78 could displace NF-Y bound to several CCAAT motifs within promoters of genes involved in cell cycle progression. DNase I footprinting of the topo IIalpha promoter confirmed binding of GWL-78 to AT-rich sequences corresponding to the preferred binding site of NF-Y. Incubation with GWL-78 resulted in displacement of NF-Y binding to DNA. Chromatin immunoprecipitation assays on the topo IIalpha promoter showed that GWL-78 was able to enter the nucleus and interact with specific DNA sequences. Treatment of NIH3T3 cells with GWL-78 resulted in a block of cell cycle progression, which did not involve activation of p53. Thus, agents such as GWL-78 may be useful in modulating transcription and blocking cellular proliferation.

Targeting the inverted CCAAT Box-2 of the topoisomerase IIalpha gene: DNA sequence selective recognition by a polyamide-intercalator as a staggered dimer.

The synthesis and DNA binding characteristics of a polyamide-intercalator conjugate, designed to inhibit NF-Y binding to the ICB-2 site of the topoisomerase IIalpha promoter and up-regulate the expression of the enzyme in confluent cells, are reported. Thermal denaturation and CD titration studies demonstrated binding to the cognate sequence (5'-AAGCTA-3'). Formation of ligand-induced CD bands at approximately 330 nm provided indication that the molecule interacts selectively in the minor groove of DNA. Intercalation was evidenced by a fivefold increase in emission of the intercalator moiety upon binding to the ICB-2 hairpin oligonucleotide. An increase in viscosity of a solution of calf-thymus DNA on addition of the conjugate provided further evidence. The binding affinity of the conjugate was ascertained using SPR (5.6x10(6) M(-1)), which according to a gel shift assay was capable of inhibiting the binding of NF-Y at a concentration of 50 microM. DNaseI footprinting, using the topoIIalpha promoter sequence, highlighted the specificity of the conjugate for the cognate site (5'-AAGCTA-3'). Finally, through Western blot analysis, confluent murine NIH 3T3 cells treated with conjugate were found to have enhanced expression of topoIIalpha. These results suggest that the conjugate can enter the nucleus, bind to its target site, presumably as a stacked dimer, and up-regulate the expression of topoIIalpha by blocking the binding of NF-Y.

Modulation of topoisomerase IIalpha expression by a DNA sequence-specific polyamide.

Topoisomerase IIalpha (topo IIalpha) is an important target for several chemotherapeutic agents, including etoposide and doxorubicin. Confluent cells express low levels of topo IIalpha and are resistant to etoposide treatment. Repression of transcription in confluent cells is mediated by binding of the transcription factor NF-Y to inverted CCAAT motifs within the topo IIalpha promoter. To block the repressive binding of NF-Y, a polyamide (JH-37) was designed to bind to the flanking regions of selected CCAAT sites within the topo IIalpha promoter. Electrophoretic mobility shift assays and DNase I footprinting assays showed occupancy of the inverted CCAAT sites by JH-37. Chromatin immunoprecipitation assays confirmed in vivo inhibition of NF-Y binding to the topo IIalpha promoter. Following incubation of confluent NIH3T3 cells with JH-37, increased expression of topo IIalpha mRNA and protein was detectable. This correlated both with increased DNA double-strand breaks as shown by comet assay and decreased cell viability following exposure to etoposide. Polyamides can modulate gene expression and chemosensitivity of cancer cells.

Binding of f-PIP, a pyrrole- and imidazole-containing triamide, to the inverted CCAAT box-2 of the topoisomerase IIalpha promoter and modulation of gene expression in cells.

An N-formamido pyrrole- and imidazole-containing triamide (f-PIP) has been shown by DNase I footprinting, SPR, and CD studies to bind as a stacked dimer to its cognate sequences: 5'-TACGAT-3' (5'-flank of the inverted CCAAT box-2 of the human topoisomerase IIalpha promoter) and 5'-ATCGAT-3'. A gel shift experiment provided evidence for f-PIP to inhibit protein-DNA interaction at the ICB2 site. Western blot studies showed that expression of the topoisomerase IIalpha gene in confluent NIH 3T3 cells was induced by treatment with f-PIP. The results suggested that the triamide was able to enter the nucleus, interacted with the target site within ICB2, inhibited NF-Y binding, and activated gene expression.

Targeting the inverted CCAAT box 2 in the topoisomerase IIalpha promoter by JH-37, an imidazole-pyrrole polyamide hairpin: design, synthesis, molecular biology, and biophysical studies.

The topoisomerase IIalpha promoter is regulated through transcription factor interactions with five inverted CCAAT boxes (ICBs). In confluent cancer cells, binding of nuclear factor Y to ICB2 represses the expression of this gene, contributing to resistance to topoisomerase II poisons. The ICB sites within the topoisomerase IIalpha promoter are, therefore, potential targets for the design of anticancer drugs and gene control agents. The synthesis and DNA binding properties of a hairpin polyamide molecule (JH-37) that targets 5'-TTGGT-3' found in ICB2 and ICB3 sites are described. Gel shift and DNase I footprinting studies on the topoisomerase IIalpha promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1 sites. The larger DeltaT(M) values for ICB2,3 (8-9 degrees C) over ICB1,4,5 (4-5 degrees C) indicated a preference of JH-37 for ICB2,3. CD titration studies confirmed the binding of JH-37 to the minor groove, with a 1:1 binding stoichiometry. Results from SPR studies showed JH-37 to bind most strongly to ICB2 (K = 3 x 10(7) M(-1)), followed by ICB1, the non-ICB sequence (TGCA), and finally the ICB mutant (ICB2m). The improved binding to ICB2 is largely due to a lower dissociation rate of the compound at the preferred site. To our knowledge, this is the first example on the use of SPR for studying the interactions of hairpin polyamides with DNA. Binding of JH-37 to ICB2 was corroborated by ITC studies, in which the DeltaG degrees of binding is driven by both enthalpy and entropy. With knowledge of the fundamental thermodynamic and kinetic properties that govern the molecular recognition of polyamides with DNA, we are poised to systematically edit the structure of JH-37 in order to further enhance its binding affinity and selectivity for ICB2,3. Our strategy for designing molecules that control gene expression is to target shorter, but multiple, binding sites that are in close array within the promoter. Binding of JH-37 to multiple ICB sites in the topoisomerase IIalpha promoter is an ideal test for this strategy. This approach is in contrast to the traditional strategy of targeting 15-16 base pairs, which has not been successful in actual biological systems due to poor cell uptake and distribution.

Enhanced tumour growth after DNA vaccination against human papilloma virus E7 oncoprotein: evidence for tumour-induced immune deviation.

We have examined the induction of anti-tumour immunity in a murine model using a gene vaccine approach to deliver a well defined tumour antigen. The vaccines expressed the human papilloma virus type 16 (HPV 16) E7 oncoprotein, and protection was measured against HPV 16-expressing C3R tumour cell line in vivo. In control mice injected with saline, C3R cells initially formed tumours but then regressed completely. As expected, animals injected with a peptide that represents the D(b)-presented CTL epitope from E7 (RAHYNIVTF) were completely protected from tumour growth. Contrary to expectation, however, we consistently saw enhanced tumour growth, delayed regression, or tumour outgrowth in mice vaccinated with two different E7-expressing DNA vaccines. We found no evidence for loss of D(b) or K(b) class I MHC molecules from C3R cells recovered from outgrown tumours, and fluorescent MHC/peptide tetramer staining revealed E7 gene vaccination did not delete RAHYNIVTF-specific CD8(+) T cells. However, we did observe an effect on cytokine production. Splenocytes from E7 gene vaccinated animals responded to re-stimulation in vitro with C3R cells by producing IL-4 but background levels of IFN-gamma. We also observed that cytokine production and E7 peptide-specific CTL were only detectable in vaccinated animals after C3R challenge, but not after DNA priming alone. We conclude that 'prime-boosting' is necessary to observe tumour-specific T cell responses with the gene vaccine approach, but that boosting with tumour cells causes skewing of the primed cells in a T2 direction that is incompatible with protective anti-tumour immunity.