Solution-phase synthesis of pyrrole-imidazole polyamides

Pyrrole−imidazole polyamides are DNA-binding molecules that are programmable for a large repertoire of DNA sequences. Typical syntheses of this class of heterocyclic oligomers rely on solid-phase methods. Solid-phase methodologies offer rapid assembly on a micromole scale sufficient for biophysical characterizations and cell culture studies. In order to produce gram-scale quantities necessary for efficacy studies in animals, polyamides must be readily synthesized in solution. An 8-ring hairpin polyamide 1, which targets the DNA sequence 5′-WGWWCW-3′, was chosen for our synthesis studies as this oligomer exhibits androgen receptor antagonism in cell culture models of prostate cancer. A convergent solution-phase synthesis of 1 from a small set of commercially available building blocks is presented which highlights principles for preparing gram quantities of pyrrole−imidazole oligomers with minimal chromatography

Oligomerization Route to Py-Im Polyamide Macrocycles

Cyclic eight-ring pyrrole−imidazole polyamides are sequence-specific DNA-binding small molecules that are cell permeable and can regulate endogenous gene expression. Syntheses of cyclic polyamides have been achieved by solid-phase and solution-phase methods. A rapid solution-phase oligomerization approach to eight-ring symmetrical cyclic polyamides yields 12- and 16-membered macrocycles as well. A preference for DNA binding by the 8- and 16-membered oligomers was observed over the 12-ring macrocycle, which we attributed to a conformational constraint not present in the smaller and larger systems

The influence of single base triplet changes on the stability of a Pur·Pur·Pyr triple helix determined by affinity cleaving

The influence of sixteen base triplet changes at a single position within a pur·pur·pyr triple helix was examined by affinity cleaving. For the 15 base pair target site studied here, G·GC, A·AT and T·AT triplets stabilize a triple helix to a greater extent than the other 13 natural triplets (pH = 7.4, 25°C). Weaker interactions were detected for the C·AT, A·GC and T·CG triplets. The absence of specific, highly stabilizing interactions between third strand bases and the CG or TA base pairs demonstrates a current sequence limitation to formation of this structure. Models for the two dimensional base triplet interactions for all possible 16 natural triplets are presented

Sequence specific suppression of androgen receptor–DNA binding in vivo by a Py-Im polyamide

The crucial role of androgen receptor (AR) in prostate cancer development is well documented, and its inhibition is a mainstay of prostate cancer treatment. Here, we analyze the perturbations to the AR cistrome caused by a minor groove binding molecule that is designed to target a sequence found in a subset of androgen response elements (ARE). We find treatment with this pyrrole-imidazole (Py-Im) polyamide exhibits sequence selectivity in its repression of AR binding in vivo. Differentially changed loci are enriched for sequences resembling ARE half-sites that match the Py-Im polyamide binding preferences determined in vitro. Comparatively, permutations of the ARE half-site bearing single or double mismatches to the Py-Im polyamide binding sequence are not enriched. This study confirms that the in vivo perturbation pattern caused by a sequence specific polyamide correlates with its in vitro binding preference genome-wide in an unbiased manner

Effects of an abasic site on triple helix formation characterized by affinity cleaving

The stability of triple helical complexes of pyrimidine oligodeoxyribonucleotides containing one abasic 1,2- dideoxy-D-ribose (ø) residue was examined by affinity cleaving. Within a pyrimidine third strand, the triplets ø·AT, ø·GC, ø·TA and ø·CG are significantly less stable than the triplets, T- AT, C + GC and GTA. The decrease in binding produced by an abasic residue is similar to that observed with imperfectly matched natural base triplets, with ø · AT and ø · GC being less stable than ø · TA and ø · CG triplets for the sequences studied

Cyclic pyrrole-imidazole polyamides targeted to the androgen response element

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 γ-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

DNA crosslinking and biological activity of a hairpin polyamide–chlorambucil conjugate

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site. Conjugate-induced lesions were first detected on DNA treated for 1 h with 0.1 muM 1-Chl, indicating that the conjugate is at least 100-fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 muM concentration. Treatment with 1-Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1-Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G(2)/M phase. Moreover, cellular effects were observed at 1-Chl concentrations similar to those needed for DNA damage in cell-free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1-Chl possesses the sequence-selectivity of a polyamide and the enhanced DNA reactivity of Chl

Tumor Xenograft Uptake of a Pyrrole−Imidazole (Py-Im) Polyamide Varies as a Function of Cell Line Grafted

Subcutaneous xenografts represent a popular approach to evaluate efficacy of prospective molecular therapeutics in vivo. In the present study, the C-14 labeled radioactive pyrrole–imidazole (Py-Im) polyamide 1, targeted to the 5′-WGWWCW-3′ DNA sequence, was evaluated with regard to its uptake properties in subcutaneous xenografts, derived from the human tumor cell lines LNCaP (prostate), A549 (lung), and U251 (brain), respectively. Significant variation in compound tumor concentrations was seen in xenografts derived from these three cell lines. Influence of cell line grafted on systemic polyamide elimination was established. With A549, a marked variation in localization of 1 was determined between Matrigel-negative and -positive xenografts. An extensive tissue distribution analysis of 1 in wild-type animals was conducted, enabling the comparison between the xenografts and the corresponding host organs of origin

Development of a novel polyamide-based agent to inhibit EVI1 function

The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME. Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor

An HRE-binding Py-Im polyamide impairs hypoxic signaling in tumors

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1 has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1 in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1 distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1 show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response