Orientation preferences of hairpin pyrrole–imidazole polyamides toward mCGG site

Hairpin pyrrole–imidazole (Py-Im) polyamides are promising medium-sized molecules that bind sequence-specifically to the minor groove of B-form DNA. Here, we synthesized a series of hairpin Py-Im polyamides and explored their binding affinities and orientation preferences to methylated DNA with the ^mCGG target sequence. Thermal denaturation assays revealed that the five hairpin Py-Im polyamides, which were anticipated to recognize ^mCGG in a forward orientation, bind to nontarget DNA, GG^mC, in a reverse orientation. Therefore, we designed five Py-Im polyamides that could recognize ^mCGG in a reverse orientation. We found that the two Py-Im polyamides containing Im/β pairs preferentially bound to ^mCGG in a reverse orientation. The reverse binding Py-Im polyamide successfully inhibited TET1 binding on the methylated DNA. Taken together, this study illustrated the importance of designing reverse binding Py-Im polyamides for the target sequence, ^mCGG, which paved the way for Py-Im polyamides that can be used with otherwise difficult to access DNA with CG sequences.     

Photocontrollable sequence-specific DNA alkylation by a pyrrole-imidazole polyamide seco-CBI conjugate

We designed and synthesized a Py-Im polyamide seco-CBI conjugate protected by a photocleavable group and demonstrated that it was selectively activated by UV irradiation both in vitro and in vivo. Sequence-specific alkylating Py-Im polyamides containing photolabile linkers may be useful for developing novel chemical- or enzyme-activated anticancer agents and may facilitate spatiotemporal control of gene expression.     

Enhancing the cellular uptake of Py-Im polyamides through next-generation aryl turns

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.     

Efficient DNA alkylation by a pyrrole-imidazole CBI conjugate with an indole linker: sequence-specific alkylation with nine-base-pair recognition

Conjugates 7, 8, and 10 of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) with a 5-amino-1H-indole-2-carbonyl linker were synthesized by Fmoc solid-phase synthesis and a subsequent liquid-phase coupling procedure. The DNA alkylating abilities of conjugates 7, 8, 6b, and 10 were examined using Texas Red-labeled PCR fragments and high-resolution denaturing gel electrophoresis. CBI conjugates 7 and 8 exhibited highly efficient sequence-specific DNA alkylation comparable with previous CBI conjugates with a vinyl linker. In particular, conjugate 10, with a 10-ringed hairpin Py-Im polyamide, alkylated at the adenine of 5'-ACAAATCCA-3'. Introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides.     

Detection of triplet repeat sequences in the double-stranded DNA using pyrene-functionalized pyrrole-imidazole polyamides with rigid linkers

Methods for sequence-specific detection in double-stranded DNA (dsDNA) are becoming increasingly useful and important as diagnostic and imaging tools. Recently, we designed and synthesized pyrrole (Py)-imidazole (Im) polyamides possessing two pyrene moieties, 1, which showed an increased excimer emission in the presence of (CAG)(12)-containing oligodeoxynucleotides (ODN) 1 and 2. In this study, we synthesized bis-pyrenyl Py-Im polyamides with rigid linkers 2, 3, and 4 to improve their fluorescence properties. Among the conjugates, 2 showed a marked increase in excimer emission, which was dependent on the concentration of the target ODN and the number of CAG repeats in the dsDNA. Unlike conjugate 1, which has flexible linkers, the excimer emission intensity of 2 was retained at over 85%, even after 4h. Py-Im polyamides have the potential to be important diagnostic molecules for detecting genetic differences between individuals.     

Optimization and validation of a high-performance liquid chromatographic method with UV detection for the determination of pyrrole-imidazole polyamides in rat plasma

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)-imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py-Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mmx15.0 cm TOSOH Co., Japan) column maintained at 40 degrees C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0-10 min, A: 100-20%, B: 0-80% linear gradient; 10-15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py-Im polyamides after intravenous injection.     

Synthesis of Lipid Derivatives of Pyrrole Polyamide and Their Biological Activity

Novel fatty acyl and phospholipid derivatives of pyrrole polyamide were synthesized. Their cytotoxicity against a cancer cell line of MT-4 cells and those infected by human immunodeficiency virus (HIV) was examined. Although no anti-HIV activity was found, their cytotoxicitty against the cancer cells was significantly enhanced by introducing a lipophilic group into the pyrrole polyamide.     

Synthesis of lipid derivatives of pyrrole polyamide and their biological activity

Novel fatty acyl and phospholipid derivatives of pyrrole polyamide were synthesized. Their cytotoxicity against a cancer cell line of MT-4 cells and those infected by human immunodeficiency virus (HIV) was examined. Although no anti-HIV activity was found, their cytotoxicitty against the cancer cells was significantly enhanced by introducing a lipophilic group into the pyrrole polyamide.     

Synthesis and Evaluation of Oligonucleotide-Conjugated Pyrrole Polyamide-2′-Deoxyguanosine Hybrids as Novel Gene Expression Control Compounds

DNA oligonucleotide-conjugated pyrrole polyamide-2′-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T_m values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.     

Synthesis and evaluation of oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids as novel gene expression control compounds

DNA oligonucleotide-conjugated pyrrole polyamide-2'-deoxyguanosine hybrids were synthesized and examined as novel gene expression control compounds. The T(m) values and circular dichroism spectral analyses showed that the oligonucleotide-conjugated hybrids possess high DNA recognition and a very high binding affinity for DNA that includes the pyrrole polyamide binding sequence.     

Nonbonded bivalence approach to cell-permeable molecules that target DNA sequences

Polyamides such as the natural antibiotic distamycin A can form binary or ternary complexes with B-DNA. The driving forces and advantages for forming the ternary complexes are not fully understood. The computational studies reported herein suggest that three- and four-ring polyamides have a propensity for forming the same dimer conformations in water as those in their ternary complexes. The pre-dimerization of a polyamide in water facilitates the formation of the ternary complex, making the polyamide more selective, and tighter binding to the minor groove whose minimal width is predetermined by the B-DNA sequence. Relative to the dimer tethered with covalent bonds, the smaller, monomeric polyamide available from reversible dimerization in water makes the molecule inherently more cell permeable. A nonbonded bivalence approach that dimerizes molecules by intermolecular interactions is proposed for improving affinity, selectivity, and cell permeability.     

DNA sequence recognition in the minor groove by hairpin pyrrole polyamide-Hoechst 33258 analogue conjugate

A hairpin pyrrole polyamide conjugated to a Hoechst 33258 (Ht) analogue, PyPyPy-gamma-PyPyPy-gamma-Ht, was synthesized on solid-phase by adaptation of an Fmoc technique using a series of PyBOP/HOBt mediated coupling reactions. Sequence selectivity and complex stabilities were characterized by spectrofluorometric titrations and thermal melting studies. The polyamide of the conjugate was observed to bind in a hairpin motif forming 1:1 conjugate:dsDNA complexes. The conjugate is able to recognize nine contiguous A/T bps, discriminating from the sequences containing fewer than nine contiguous A/T bps.     

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.     

Modulation of the EMT/MET process by pyrrole–imidazole polyamide targeting human transforming growth factor-β1

Transforming growth factor-β1 (TGF-β1) is a potent induction factor for epithelial–mesenchymal transition (EMT). Mesenchymal–epithelial transition (MET), as the inverse process of EMT, has recently been reported to promote the induction of induced pluripotent stem cells (iPSCs). We have developed pyrrole–imidazole (PI) polyamide, a novel gene regulator that targets human TGF-β1, and investigated its effects on the EMT/MET process. PI polyamide targeted to TGF-β1 significantly inhibited the mRNA expression of TGF-β1 and SNAI1 as an EMT marker and increased mRNA and protein expression of E-cadherin in human epithelial cells. To enhance the induction of iPSCs by the MET process, PI polyamide targeted to TGF-β1 was applied to human fibroblasts transfected with exogenous reprogramming factors by Sendai virus vector and grown in human iPSCs. The PI polyamide significantly increased the number of alkaline phosphatase-positive colonies. The expression of undifferentiated markers was also observed in these colonies. These results suggest that PI polyamide targeted to human TGF-β is a novel compound that can control the EMT/MET process of human epithelial cells and enhance the induction of human fibroblasts to iPSCs.     

Novel diamino imidazole and pyrrole-containing polyamides: Synthesis and DNA binding studies of mono- and diamino-phenyl-ImPy*Im polyamides designed to target 5'-ACGCGT-3'

Pyrrole- and imidazole-containing polyamides are widely investigated as DNA sequence selective binding agents that have potential use as gene control agents. The key challenges that must be overcome to realize this goal is the development of polyamides with low molar mass so the molecules can readily diffuse into cells and concentrate in the nucleus. In addition, the molecules must have appreciable water solubility, bind DNA sequence specifically, and with high affinity. It is on this basis that the orthogonally positioned diamino/dicationic polyamide Ph-ImPy*Im 5 was designed to target the sequence 5'-ACGCGT-3'. Py* denotes the pyrrole unit that contains a N-substituted aminopropyl pendant group. The DNA binding properties of diamino polyamide 5 were determined using a number of techniques including CD, ΔT(M), DNase I footprinting, SPR and ITC studies. The effects of the second amino moiety in Py* on DNA binding affinity over its monoamino counterpart Ph-ImPyIm 3 were assessed by conducting DNA binding studies of 3 in parallel with 5. The results confirmed the minor groove binding and selectivity of both polyamides for the cognate sequence 5'-ACGCGT-3'. The diamino/dicationic polyamide 5 showed enhanced binding affinity and higher solubility in aqueous media over its monoamino/monocationic counterpart Ph-ImPyIm 3. The binding constant of 5, determined from SPR studies, was found to be 1.5 × 10(7)M(-1), which is ～3 times higher than that for its monoamino analog 3 (4.8 × 10(6)M(-1)). The affinity of 5 is now approaching that of the parent compound f-ImPyIm 1 and its diamino equivalent 4. The advantages of the design of diamino polyamide 5 over 1 and 4 are its sequence specificity and the ease of synthesis compared to the N-terminus pyrrole analog 2.     

Synthesis,reactivity and biological evaluation of novel halogenated tripentones

We describe herein the synthesis and the biological evaluation of a novel series of a potent anticancer agents: the tripentones. For the first time, a halogen atom was introduced in high yields on the pyrrole ring of the tricycle. This synthesis and the reactivity of the novel halogenated tripentones in metallo-catalysed cross-coupling reactions will be described in that article. Finally their influence on biological activity will be discussed.     

Synthesis and DNA binding properties of 1-(3-aminopropyl)-imidazole-containing triamide f-Im(∗)PyIm: A novel diamino polyamide designed to target 5'-ACGCGT-3'

A novel diamino/dicationic polyamide f-Im(?)PyIm (5) that contains an orthogonally positioned aminopropyl chain on an imidazole (Im(?)) moiety was designed to target 5'-ACGCGT-3'. The DNA binding properties of the diamino polyamide 5, determined by CD, ΔT(M), DNase I footprinting, SPR, and ITC studies, were compared with those of its monoamino/monocationic counterpart f-ImPyIm (1) and its diamino/dicationic isomer f-ImPy(?)Im (2), which has the aminopropyl group attached to the central pyrrole unit (Py(?)). The results gave evidence for the minor groove binding and selectivity of polyamide 5 for the cognate sequence 5'-ACGCGT-3', and with strong affinity (K(eq)=2.3×10(7)M(-1)). However, the binding affinities varied according to the order: f-ImPy(?)Im (2)>f-ImPyIm (1)?f-Im(?)PyIm (5) confirming that the second amino group can improve affinity, but its position within the polyamide can affect affinity.