Biomedical subjects

Peter B Dervan

Publications and source records attributed to Peter B Dervan.

At least 19 recordsLinked to original sources

PubMed2006

Unanticipated differences between alpha- and gamma-diaminobutyric acid-linked hairpin polyamide-alkylator conjugates.

Hairpin polyamide-chlorambucil conjugates containing an alpha-diaminobutyric acid (alpha-DABA) turn moiety are compared to their constitutional isomers containing the well-characterized gamma-DABA turn. Although the DNA-binding properties of unconjugated polyamides are similar, the alpha-DABA conjugates display increased alkylation specificity and decreased rate of reaction. Treatment of a human colon carcinoma cell line with alpha-DABA versus gamma-DABA hairpin conjugates shows only slight differences in toxicities while producing similar effects on cell morphology and G2/M stage cell cycle arrest. However, striking differences in animal toxicity between the two classes are observed. Although mice treated with an alpha-DABA hairpin polyamide do not differ significantly from control mice, the analogous gamma-DABA hairpin is lethal. This dramatic difference from a subtle structural change would not have been predicted.

Alkylating Agents↗

PubMed2006

Improved nuclear localization of DNA-binding polyamides.

Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus 'tail' has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.

Binding Sites↗

PubMed2006

Programmable oligomers targeting 5'-GGGG-3' in the minor groove of DNA and NF-kappaB binding inhibition.

A series of hairpin oligomers containing benzimidazole (Bi) and imidazopyridine (Ip) rings were synthesized and screened to target 5'-WGGGGW-3', a core sequence in the DNA-binding site of NF-kappaB, a prolific transcription factor important in biology and disease. Five Bi and Ip containing oligomers bound to the 5'-WGGGGW-3' site with high affinity. One of the oligomers (Im-Im-Im-Im-gamma-Py-Bi-Py-Bi-beta-Dp) was able to inhibit DNA binding by the transcription factor NF-kappaB.

Base Sequence↗

PubMed2006

Exploring the limits of benzimidazole DNA-binding oligomers for the hypoxia inducible factor (HIF) site.

The vascular endothelial growth factor (VEGF) and its receptors have been implicated as key-factors in tumor angiogenesis and are major targets in cancer therapy. New oligomers which mimic the architecture of DNA-binding polyamides have been designed to target the hypoxia inducible factor (HIF-1alpha) binding site on the promoter of VEGF gene. These oligomers incorporate an increasing number of six-five fused rings such as hydroxybenzimidazole-imidazole, benzimidazole-pyrrole, benzimidazole-chlorothiophene, and imidazopyridine-pyrrole, and bind the VEGF hypoxia response element (HRE) 5'-TACGT-3' with high affinity and selectivity.

Benzimidazoles↗

PubMed2006

DNA sequence-specific polyamides alleviate transcription inhibition associated with long GAA.TTC repeats in Friedreich's ataxia.

The DNA abnormality found in 98% of Friedreich's ataxia (FRDA) patients is the unstable hyperexpansion of a GAA.TTC triplet repeat in the first intron of the frataxin gene. Expanded GAA.TTC repeats result in decreased transcription and reduced levels of frataxin protein in affected individuals. Beta-alanine-linked pyrrole-imidazole polyamides bind GAA.TTC tracts with high affinity and disrupt the intramolecular DNA.DNA-associated region of the sticky-DNA conformation formed by long GAA.TTC repeats. Fluorescent polyamide-Bodipy conjugates localize in the nucleus of a lymphoid cell line derived from a FRDA patient. The synthetic ligands increase transcription of the frataxin gene in cell culture, resulting in increased levels of frataxin protein. DNA microarray analyses indicate that a limited number of genes are significantly affected in FRDA cells. Polyamides may increase transcription by altering the DNA conformation of genes harboring long GAA.TTC repeats or by chromatin opening.

Cell Line↗

PubMed2006

Programmable oligomers for minor groove DNA recognition.

The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.

Base Pairing↗

PubMed2006

A two-hit mechanism for pre-mitotic arrest of cancer cell proliferation by a polyamide-alkylator conjugate.

A polyamide-chlorambucil conjugate (1R-Chl) arrests a wide range of human cancer cell lines at the G2/M phase of the cell cycle and downregulates histone H4c gene expression. However, an siRNA against H4c mRNA causes G1/S arrest. Here, we report that 1R-Chl downregulates H4c prior to G2/M arrest. G2/M arrest is the result of extensive DNA damage by 1R-Chl, which leads to phosphorylation of H2A.X at serine 139, recruitment of the Nbs1 repair protein, and a cascade of unknown events culminating with cdc2 phosphorylation at tyrosine 15 and abolishment of cdc2 kinase activity. A control polyamide-Chl conjugate, which neither binds to the H4c gene nor has an anti-proliferative effect by itself, causes G2/M arrest when cells are treated with siRNAs specific for H3 or H4c.

Cell Division↗

PubMed2006

Defining the sequence-recognition profile of DNA-binding molecules.

Determining the sequence-recognition properties of DNA-binding proteins and small molecules remains a major challenge. To address this need, we have developed a high-throughput approach that provides a comprehensive profile of the binding properties of DNA-binding molecules. The approach is based on displaying every permutation of a duplex DNA sequence (up to 10 positional variants) on a microfabricated array. The entire sequence space is interrogated simultaneously, and the affinity of a DNA-binding molecule for every sequence is obtained in a rapid, unbiased, and unsupervised manner. Using this platform, we have determined the full molecular recognition profile of an engineered small molecule and a eukaryotic transcription factor. The approach also yielded unique insights into the altered sequence-recognition landscapes as a result of cooperative assembly of DNA-binding molecules in a ternary complex. Solution studies strongly corroborated the sequence preferences identified by the array analysis.

Animals↗

PubMed2005

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates.

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.

Base Sequence↗

PubMed2005

Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor alpha with minor groove binding polyamides.

Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.

Binding, Competitive↗

PubMed2005

Temperature-sensitive protein-DNA dimerizers.

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a "switch"-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry.

Amino Acid Sequence↗

PubMed2005

Nucleosomes in solution exist as a mixture of twist-defect states.

The 2.0 A crystal structure of a nucleosome core particle in complex with a bivalent pyrrole-imidazole polyamide reveals that this "clamp" effectively crossbraces the two gyres of the DNA superhelix, thereby stabilizing the nucleosome against dissociation. Using X-ray crystallography and footprinting techniques, we show that the clamp preferentially binds nucleosomes over free DNA, and that nucleosomal DNA exists as a mixture of multiple twist-defect intermediates in solution. The nucleosomes exist in one of two different conformations in various crystal structures that trap twist-defect intermediates, even on a strong positioning sequence. Evidence has been obtained supporting the existence of twist-defect states in nucleosomal DNA in solution that are similar to those obtained in crystal structures. Our results also substantiate the idea that twist diffusion may represent an important means of altering the accessibility of nucleosomal DNA both in the presence and in the absence of ATP-dependent chromatin-remodelling enzymes.

Animals↗

PubMed2005

Programmable DNA binding oligomers for control of transcription.

Mapping and sequencing the genetic blueprint in human, mice, yeast and other model organisms has created challenges and opportunities for chemistry, biology and human medicine. An understanding of the function of each of the approximately 25,000 genes in humans, and the biological circuitry that controls these genes will be driven in part by new technologies from the world of chemistry. Many cellular events that lead to cancer and the progression of human disease represent aberrant gene expression. Small molecules that can be programmed to mimic transcription factors and bind a large repertoire of DNA sequences in the human genome would be useful tools in biology and potentially in human medicine. Polyamides are synthetic oligomers programmed to read the DNA double helix. They are cell permeable, bind chromatin and have been shown to downregulate endogenous genes in cell culture.

Animals↗

PubMed2004

Small molecule transcription factor mimic.

Regulation of gene expression by transcription factors touches many aspects of eukaryotic biology, and its systematic, external control by organic molecules represents a challenge in chemistry. Here we report the design of a completely organic, nonpeptidic compound that mimics a transcription factor. The design takes advantage of the specific DNA-binding affinity of a hairpin polyamide molecule and the ability of wrenchnolol to bind to the Sur-2 subunit of human mediator complex. The hybrid compound of these two molecules activates transcription of a reporter gene in vitro in a promoter-dependent manner through simultaneous contacts with DNA and Sur-2. Our results indicate that it is possible to generate a transcription factor out of nonpeptidic components.

Adamantane↗

PubMed2004

DNA damage effects of a polyamide-CBI conjugate in SV40 virions.

Polyamides are a class of synthetic molecules that exhibit high-affinity, sequence-specific reversible binding in the DNA minor groove but are incapable of inducing DNA damage. In cell-free systems, polyamides have been shown to regulate gene expression by activation, repression, and antirepression. However, effectiveness in cell culture has met with limited success and seems to be cell-dependent. By combining a polyamide with a moiety of a DNA-alkylating agent of the cyclopropylpyrroloindole (CPI) family, a conjugate molecule [polyamide 1-CBI (1-(chloromethyl)-5-hydroxyl-1,2-dihydro-3H-benz[e]indole) conjugate] capable of sequence-specific DNA alkylation was shown to exhibit cellular activity (i.e., cell-growth inhibition and cell-cycle arrest) in mammalian cells. These effects, however, occur at concentrations several orders of magnitude higher than those of its parent CPI agent adozelesin. In addition, 1-CBI is able to interact sequence-specifically with viral DNA and inhibit SV40 DNA replication in infected BSC-1 (African green monkey kidney epithelial) cells, albeit at a greatly reduced ability compared with its CPI parent. On the basis of results from previous studies, we tested whether pretreatment of virus with 1-CBI, compared with direct treatment of infected cells, would enhance its cellular activity. Therefore, using SV40 virions as a model system, we examined the ability of this conjugate molecule to penetrate SV40 virions and damage viral DNA. Our results demonstrate that 1-CBI is able to damage encapsidated SV40 DNA. Both DNA replication and virus production are effectively inhibited in a concentration-dependent manner after infection of BSC-1 cells with 1-CBI-pretreated virions. It is surprising that, unlike in mammalian cells, the relative activity of 1-CBI in SV40 virions is comparable with that of the highly cytotoxic CPI agent adozelesin. Because 1-CBI is able to efficiently penetrate virions and damage DNA, these findings may provide the framework for the development of polyamide-based antiviral agents with enhanced sequence-preference capabilities.

DNA Damage↗

PubMed2004

Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist.

Vascular endothelial growth factor (VEGF) and its receptors have been implicated as key factors in tumor angiogenesis that are up-regulated by hypoxia. We evaluated the effects of DNA-binding small molecules on hypoxia-inducible transcription of VEGF. A synthetic pyrrole-imidazole polyamide designed to bind the hypoxia response element (HRE) was found to disrupt hypoxia-inducible factor (HIF) binding to HRE. In cultured HeLa cells, this resulted in a reduction of VEGF mRNA and secreted protein levels. The observed effects were polyamide-specific and dose-dependent. Analysis of genome-wide effects of the HRE-specific polyamide revealed that a number of hypoxia-inducible genes were down-regulated. Pathway-based regulation of hypoxia-inducible gene expression with DNA-binding small molecules may represent a new approach for targeting angiogenesis.

Base Sequence↗

PubMed2004

Polyamides reveal a role for repression in latency within resting T cells of HIV-infected donors.

BACKGROUND: The persistence of human immunodeficiency virus (HIV) type 1 within resting CD4+ T cells poses a daunting therapeutic challenge. Histone deacetylase (HDAC)-1, a chromatin-remodeling enzyme that can mediate gene silencing, is recruited to the HIV-1 long terminal repeat by the host transcription factor LSF. Pyrrole-imidazole polyamides, small molecules that target specific DNA sequences, can access the nucleus of cells and specifically block transcription-factor binding. METHODS: We used polyamides to directly test the role of chromatin remodeling in HIV quiescence in primary resting CD4+ T cells obtained from HIV-infected patients. RESULTS: After exposure to any of 4 different polyamides that specifically block HDAC-1 recruitment by LSF to the HIV promoter, replication-competent HIV was recovered from cultures of resting CD4+ T cells in 6 of 8 HIV-infected patients whose viremia had been suppressed by therapy. In comparison, HIV was not recovered after exposure to control, mismatched polyamides but was recovered from 7 of 8 of these patients' samples after the activation of T cells. CONCLUSIONS: We identify histone deacetylation as a mechanism that can dampen viral expression in infected, activated CD4+ T cells and establish a persistent, quiescent reservoir of HIV infection.

Anti-HIV Agents↗

PubMed2004

Paradoxical effects of DNA binding polyamides on HTLV-1 transcription.

Human T-cell leukemia virus type-1 (HTLV-1) depends on the virally encoded transcription factor Tax for efficient viral replication and gene expression. In a complex with CREB, Tax contacts the minor groove of the promoter DNA at guanine and cytosine rich sequences that flank three of the off-consensus cyclic-AMP response elements (CREs). In this study, we used six Tax-directed pyrrole-imidazole polyamides specifically designed to block Tax binding to DNA at each GC sequence of the three viral CREs. We found that four of these polyamides disrupt binding of the Tax/CREB complex in vitro, and that these same molecules also inhibit Tax-mediated transcription in vitro on chromatin-assembled templates. However, of these four Tax/CREB-specific polyamides, only one polyamide appears to be uniquely Tax specific. We show that polyamides can enter the nuclei of HTLV-1 infected T-cells, and two of the four polyamides down-regulated virion production in these cells. Together, these data illustrate the importance of studying polyamide inhibition of gene expression in vitro and in vivo, as the function of the polyamides in living cells is not fully understood. Finally, our data indicates that targeted disruption of the Tax/CREB complex, or other complexes which assemble on the HTLV-1 promoter, may provide a novel approach for inhibiting viral replication in vivo.

Animals↗