Examining NTA performance and potential using fortified and reference house dust as part of EPA's Non-Targeted Analysis Collaborative Trial (ENTACT).

Non-targeted analysis (NTA) methods are being increasingly used to aid in the identification of unknown compounds in the environment, a problem that has challenged environmental chemists for decades. Despite its increased use, quality assurance practices for NTA have not been well established. Furthermore, capabilities and limitations of certain NTA methods have not been thoroughly evaluated. Standard reference material dust (SRM 2585) was used here to evaluate the ability of NTA to identify previously reported compounds, as well as a suite of 365 chemicals that were spiked at various stages of the analytical procedure. Analysis of the unaltered SRM 2585 extracts revealed that several previously reported compounds can be identified by NTA, and that correct identification was dependent on concentration. A manual inspection of unknown features in SRM 2585 revealed the presence of two chlorinated and fluorinated compounds in high abundance, likely precursors to perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS). A retrospective analysis of data from the American Healthy Homes Survey revealed that these compounds were present in 42% of sampled homes. Spiking the dust at various stages of sample preparation revealed losses from extraction, cleanup, and instrumental analysis; the log Kow for individual compounds influenced the overall recovery levels but no pattern could be discerned from the various degrees of interference that the matrix had on the ionization efficiency of the spiked chemicals. Analysis of the matrix-free chemical mixture at low, medium, and high concentrations led to more correct identifications than analysis at one, very high concentration. Varying the spiked amount and identifying reported compounds at known concentrations allowed an estimation of the lower limits of identification (LOIs) for NTA, analogous to limits of detection in targeted analysis. The LOIs were much lower than levels in dust that would be likely to cause bioactivity in humans, indicating that NTA is useful for identifying and monitoring compounds that may be of toxicological concern. Graphical abstract.

Small molecule binders recognize DNA microstructural variations via an induced fit mechanism.

Regulation of gene-expression by specific targeting of protein-nucleic acid interactions has been a long-standing goal in medicinal chemistry. Transcription factors are considered "undruggable" because they lack binding sites well suited for binding small-molecules. In order to overcome this obstacle, we are interested in designing small molecules that bind to the corresponding promoter sequences and either prevent or modulate transcription factor association via an allosteric mechanism. To achieve this, we must design small molecules that are both sequence-specific and able to target G/C base pair sites. A thorough understanding of the relationship between binding affinity and the structural aspects of the small molecule-DNA complex would greatly aid in rational design of such compounds. Here we present a comprehensive analysis of sequence-specific DNA association of a synthetic minor groove binder using long timescale molecular dynamics. We show how binding selectivity arises from a combination of structural factors. Our results provide a framework for the rational design and optimization of synthetic small molecules in order to improve site-specific targeting of DNA for therapeutic uses in the design of selective DNA binders targeting transcription regulation.

Targeting Multiple Effector Pathways in Pancreatic Ductal Adenocarcinoma with a G-Quadruplex-Binding Small Molecule.

Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[ lmn][3,8]phenanthroline-1,3,6,8(2 H,7 H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers.

DNA microstructure influences selective binding of small molecules designed to target mixed-site DNA sequences.

Specific targeting of protein­nucleic acid interactions is an area of current interest, for example, in the regulation of gene-expression. Most transcription factor proteins bind in the DNA major groove; however, we are interested in an approach using small molecules to target the minor groove to control expression by an allosteric mechanism. In an effort to broaden sequence recognition of DNA-targeted-small-molecules to include both A·T and G·C base pairs, we recently discovered that the heterocyclic diamidine, DB2277, forms a strong monomer complex with a DNA sequence containing 5΄-AAAGTTT-3΄. Competition mass spectrometry and surface plasmon resonance identified new monomer complexes, as well as unexpected binding of two DB2277 with certain sequences. Inherent microstructural differences within the experimental DNAs were identified through computational analyses to understand the molecular basis for recognition. These findings emphasize the critical nature of the DNA minor groove microstructure for sequence-specific recognition and offer new avenues to design synthetic small molecules for effective regulation of gene-expression.

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide.

BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2µM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.