Cyclic Pyrrole-Imidazole Polyamides Targeted to the Androgen Response Element

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 06/2009; 131(20):7182-8. DOI: 10.1021/ja901309z
Source: PubMed


Hairpin pyrrole-imidazole (Py-Im) polyamides are a class of cell-permeable DNA-binding small molecules that can disrupt transcription factor-DNA binding and regulate endogenous gene expression. The covalent linkage of antiparallel Py-Im ring pairs with an gamma-amino acid turn unit affords the classical hairpin Py-Im polyamide structure. Closing the hairpin with a second turn unit yields a cyclic polyamide, a lesser-studied architecture mainly attributable to synthetic inaccessibility. We have applied our methodology for solution-phase polyamide synthesis to cyclic polyamides with an improved high-yield cyclization step. Cyclic 8-ring Py-Im polyamides 1-3 target the DNA sequence 5'-WGWWCW-3', which corresponds to the androgen response element (ARE) bound by the androgen receptor transcription factor to modulate gene expression. We find that cyclic Py-Im polyamides 1-3 bind DNA with exceptionally high affinities and regulate the expression of AR target genes in cell culture studies, from which we infer that the cycle is cell permeable.

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Available from: David M Chenoweth, Apr 03, 2014
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    • "The low amount of renal and biliary elimination of compound 1 may be suggestive of compound retention in the tissues or its metabolic degradation. A previous absorption, distribution, metabolism, excretion, and toxicity (ADMET) study had ascertained that polyamide 2 was resistant to liver microsomal degradation [5]; however, the microsomal stability of polyamide 1 was never examined, and thus, enzymatic degradation could be a route of elimination for this compound. "
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    ABSTRACT: Pyrrole-imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove-binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide. The biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice. The variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models.
    Cancer Chemotherapy and Pharmacology 08/2012; 70(4):617-25. DOI:10.1007/s00280-012-1954-3 · 2.77 Impact Factor
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    • "Structural studies suggest this is due to the unique stereochemical presentation of the chiral β-amino moiety, which is directed up and out of the minor groove floor, thereby providing a chemical handle for introduction of functionality at the turn position (18). Furthermore, biological evaluation of a β-acetylated polyamide targeted to the consensus androgen response element (ARE) half site 5′-WGWWCW-3′ (W = A/T) showed inhibition of prostate specific antigen (PSA) gene expression at 10-fold lower concentrations relative to polyamides bearing unsubstituted β-amino turns, illustrating the ability of turn substitution to potentiate gene regulatory effects (19). "
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    ABSTRACT: Pyrrole-imidazole (Py-Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein-DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5'-WGWWCW-3' (W =A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py-Im polyamides that will prove valuable for future applications in which biological potency is essential.
    Nucleic Acids Research 11/2011; 40(5):2345-56. DOI:10.1093/nar/gkr970 · 9.11 Impact Factor
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    • "Compounds that bind in the minor groove are of current research interest for their therapeutic potential against diseases from cancer to a variety of infectious diseases and for biotechnology applications (1–15). For example, the DNA minor groove continues to be an important target for the development of anti-cancer (1–5,15), anti-microbial and anti-viral compounds (6–9,12,14). "
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    ABSTRACT: Structural results with minor groove binding agents, such as netropsin, have provided detailed, atomic level views of DNA molecular recognition. Solution studies, however, indicate that there is complexity in the binding of minor groove agents to a single site. Netropsin, for example, has two DNA binding enthalpies in isothermal titration calorimetry (ITC) experiments that indicate the compound simultaneously forms two thermodynamically different complexes at a single AATT site. Two proposals for the origin of this unusual observation have been developed: (i) two different bound species of netropsin at single binding sites and (ii) a netropsin induced DNA hairpin to duplex transition. To develop a better understanding of DNA recognition complexity, the two proposals have been tested with several DNAs and the methods of mass spectrometry (MS), polyacrylamide gel electrophoresis (PAGE) and nuclear magnetic resonance spectroscopy in addition to ITC. All of the methods with all of the DNAs investigated clearly shows that netropsin forms two different complexes at AATT sites, and that the proposal for an induced hairpin to duplex transition in this system is incorrect.
    Nucleic Acids Research 09/2011; 39(22):9649-58. DOI:10.1093/nar/gkr699 · 9.11 Impact Factor
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