Jevgenij A. Raskatov

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5′-WGGWWW-3′ and 5′GGGWWW-3′. The compound is capable of binding to κB sites and reducing the expression of various NF-κB–driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α–inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists.

Ir-Catalysed Asymmetric Allylic Substitutions with Cyclometalated (Phosphoramidite)Ir Complexes—Resting States, Catalytically Active (π-Allyl)Ir Complexes and Computational Exploration

Mechanistic aspects of allylic substitutions with iridium catalysts derived from phosphoramidites by cyclometalation were investigated. The determination of resting states by (31)P NMR spectroscopy led to the conclusion that the cyclometalation process is reversible. A novel, one-pot procedure for the preparation of (pi- allyl)Ir complexes was developed, and these complexes were characterised by X-ray crystal structure analyses and spectral data. They are fully active catalysts of the allylic substitution reaction. DFT calculations on the allyl complexes, transition states of the allylic substitution and product olefin complexes gave further mechanistic insight.

Gene expression changes in a tumor xenograft by a pyrrole-imidazole polyamide

Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5′-WGGWWW-3′ with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. An FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which was interrogated by quantitative reverse transcription-PCR and correlated with cell culture expression levels.

Activity of a Py–Im Polyamide Targeted to the Estrogen Response Element

Pyrrole-imidazole (Py–Im) polyamides are a class of programmable DNA minor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERα) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in a majority of breast cancers. We tested a selection of structurally similar Py–Im polyamides with differing DNA sequence specificity for activity against 17β-estadiol (E2)–induced transcription and cytotoxicity in ERα positive, E2-stimulated T47DKBluc cells, which express luciferase under ERα control. The most active polyamide targeted the sequence 5′-WGGWCW-3′ (W = A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2-stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity. Mol Cancer Ther; 12(5); 675–84. ©2013 AACR.

Determination of the Conformation of the Key Intermediate in an Enantioselective Palladium-Catalyzed Allylic Substitution from Residual Dipolar Couplings†

Transition-metal-catalyzed enantioselective C C bond-forming reactions such as allylic substitution are of great importance in academic research and industrial processes. In investigating the origin of the stereoselection of such a reaction knowledge about the spatial relationships between the ligand(s), the transition metal, and the substrate is a crucial step. If an intermediate can be isolated, the spatial structure of the intermediate can in principle be obtained either by X-ray crystallography or NMR spectroscopy. However, it should be pointed out here that the equilibrium conformation of the intermediate does not necessarily lead to conclusive insights on reactivity and selectivity, since that depends on whether an early or late transition state is involved in the formation of the product. 4] Extensive studies have been devoted to probing the origin of stereoselection in allylic substitutions with bidentate ligands, of which only few can be mentioned here. For systems with monodentate ligands, as is the case in the present study (see 1), several X-ray structures of catalyst–substrate complexes exist, but to our knowledge no detailed NMR spectroscopic study in solution has yet been performed. The conformation of an intermediate complex in solution might not necessarily resemble that determined crystallographically. Furthermore, the solution conformation and in particular the dynamics of the intermediate species might be important for insight into the stereoselective step. Unfortunately, determining the predominant conformation in solution is not always straightforward, especially if organometallic species are considered. Conventional NMR parameters such as J couplings and NOEs might fail either because of missing links between spins (for both J couplings and NOEs), missing parametrizations (for J couplings especially for organometallic species), or conformational flexibility. This was also the case for the complex described herein. In contrast to the short-range information content of J couplings and NOEs, residual dipolar couplings (RDCs) provide long-range structural information and can also be used to relate non-interacting spins in biomolecular and recently also in organic compounds. The complex investigated here by means of RDCs was reported by Helmchen et al. to be a self-organizing palladium catalyst system bearing two monodentate ligands 1 and showing high activity and enantioselectivity in allylic alkylation reactions (Scheme 1). As the ligands are monodentate, considerable flexibility in the corresponding substrate– catalyst complex can be expected. It is thus very important to obtain information about the conformational behavior of the catalyst–substrate complex 2 in solution.

Characterization and Solubilization of Pyrrole−Imidazole Polyamide Aggregates

To optimize the biological activity of pyrrole–imidazole polyamide DNA-binding molecules, we characterized the aggregation propensity of these compounds through dynamic light scattering and fractional solubility analysis. Nearly all studied polyamides were found to form measurable particles 50–500 nm in size under biologically relevant conditions, while HPLC-based analyses revealed solubility trends in both core sequences and peripheral substituents that did not correlate with overall ionic charge. The solubility of both hairpin and cyclic polyamides was increased upon addition of carbohydrate solubilizing agents, in particular, 2-hydroxypropyl-β-cyclodextrin (HpβCD). In mice, the use of HpβCD allowed for improved injection conditions and subsequent investigations of the availability of polyamides in mouse plasma to human cells. The results of these studies will influence the further design of Py-Im polyamides and facilitate their study in animal models.

Iridium-catalyzed allylic substitutions with cyclometalated phosphoramidite complexes bearing a dibenzocyclooctatetraene ligand: preparation of (π-allyl)Ir complexes and computational and NMR spectroscopic studies.

(π-Allyl)Ir complexes derived from dibenzocyclooctatetraene and phosphoramidites by cyclometalation are effective catalysts for allylic substitution reactions of linear monosubstituted allylic carbonates. These catalysts provide exceptionally high degrees of regioselectivity and allow the reactions to be run under aerobic conditions. A series of (π-allyl)Ir complexes were prepared and characterized by X-ray crystal structure analyses. An allylic amination with aniline displayed different resting states depending on the presence of a strong base. DFT calculations were carried out on the mechanistic aspects of these reactions. The results suggest that for the (π-allyl)Ir complexes, the formation and reactions with nucleophiles proceed with comparable rates.

Expanding the Repertoire of Natural Product-Inspired Ring Pairs for Molecular Recognition of DNA

A furan amino acid, inspired by the recently discovered proximicin natural products, was incorporated into the scaffold of a DNA-binding hairpin polyamide. While unpaired oligomers of 2,4-disubstituted furan amino acids show poor DNA-binding activity, furan (Fn) carboxamides paired with N-methylpyrrole (Py) and N-methylimidazole (Im) rings demonstrate excellent stabilization of duplex DNA as well as discrimination of noncognate sequences, consistent with function as a Py mimic according to the Py/Im polyamide pairing rules.

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.

A C-14 labeled Py–Im polyamide localizes to a subcutaneous prostate cancer tumor

In an effort to quantitate Py-Im polyamide concentrations in vivo, we synthesized the C-14 radioactively labeled compounds 1-3, and investigated their tumor localization in a subcutaneous xenograft model of prostate cancer (LNCaP). Tumor concentrations were compared with representative host tissues, and exhibited a certain degree of preferential localization to the xenograft. Compound accumulation upon repeated administration was measured. Py-Im polyamide 1 was found to accumulate in LNCaP tumors at concentrations similar to the IC50 value for this compound in cell culture experiments.