Cyclopentane-Peptide Nucleic Acids for Qualitative, Quantitative, and Repetitive Detection of Nucleic Acids

We report the development of chemically modified peptide nucleic acids (PNAs) as probes for qualitative and quantitative detection of DNA. The remarkable stability of PNAs toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device that can be used outside of a laboratory setting. Using an enzyme-linked reporter assay, we demonstrate that excellent levels of detection and accuracy for anthrax DNA can be achieved using PNA probes with suitable chemical components designed into the probe. In addition, we report on DNA-templated cross-linking of PNA probes as a way to preserve genetic information for repetitive and subsequent analysis. This report is the first detailed examination of the qualitative and quantitative properties of chemically modified PNA for nucleic acid detection and provides a platform for studying and optimizing PNA probes prior to incorporation into new technological platforms

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain – NF54-WT or mutant strain – NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions

PNA-Based Multivalent Scaffolds Activate the Dopamine D₂ Receptor

Peptide nucleic acid scaffolds represent a promising tool to interrogate the multivalent effects of ligand binding to a membrane receptor. Dopamine D₂ receptors (D₂R) are a class of G-protein coupled receptors (GPCRs), and the formation of higher-ordered structures of these receptors has been associated with the progression of several neurological diseases. In this Letter, we describe the synthesis of a library of ligand-modified PNAs bearing a known D₂R agonist, (±)-PPHT. The D₂R activity for each construct was assessed, and the multivalent effects were evaluated

Click Dimers To Target HIV TAR RNA Conformation

A series of neomycin dimers have been synthesized using “click chemistry” with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem–loop structure located at the 5′-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (<i>T</i>_m) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC₅₀ values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity

Programmable Nanoscaffolds That Control Ligand Display to a G‑Protein-Coupled Receptor in Membranes To Allow Dissection of Multivalent Effects

A programmable ligand display system can be used to dissect the multivalent effects of ligand binding to a membrane receptor. An antagonist of the A_2A adenosine receptor, a G-protein-coupled receptor that is a drug target for neurodegenerative conditions, was displayed in 35 different multivalent configurations, and binding to A_2A was determined. A theoretical model based on statistical mechanics was developed to interpret the binding data, suggesting the importance of receptor dimers. Using this model, extended multivalent arrangements of ligands were constructed with progressive improvements in binding to A_2A. The results highlight the ability to use a highly controllable multivalent approach to determine optimal ligand valency and spacing that can be subsequently optimized for binding to a membrane receptor. Models explaining the multivalent binding data are also presented

Discovery of Novel Small-Molecule Scaffolds for the Inhibition and Activation of WIP1 Phosphatase from a RapidFire Mass Spectrometry High-Throughput Screen

Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102 277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator