Marina Tanasova

Sensing remote chirality: stereochemical determination of β-, γ-, and δ-chiral carboxylic acids.

Determining the absolute stereochemisty of small molecules bearing remote nonfunctionalizable stereocenters is a challenging task. Presented is a solution in which appropriately substituted bis(porphyrin) tweezers are used. Complexation of a suitably derivatized β-, γ-, or δ-chiral carboxylic acid to the tweezer induces a predictable helicity of the bis(porphyrin), which is detected as a bisignate Cotton Effect (ECCD). The sign of the ECCD curve is correlated with the absolute stereochemistry of the substrate based on the derived working mnemonics in a predictable manner.

Fluorescent THF‐Based Fructose Analogue Exhibits Fructose‐Dependent Uptake

Recent publications suggest that high dietary fructose might play a significant role in cancer metabolism and can exacerbate a number of aspects of metabolic syndrome. Addressing the role that fructose plays in human health is a controversial question and requires a detailed understanding of many factors including the mechanism of fructose transport into healthy and diseased cells. Fructose transport into cells is thought to be largely mediated by the passive hexose transporters Glut2 and Glut5. To date, no probes that can be selectively transported by one of these enzymes but not by the other have been identified. The data presented here indicate that, in MCF‐7 cells, a 1‐amino‐2,5‐anhydro‐D‐mannitol‐based fluorescent NBDM probe is transported twice as efficiently as fructose and that this takes place with the aid of Glut5. Its Glut5 specificity and differential uptake in cancer cells and in normal cells suggest this NBDM probe as a potentially useful tool for cross‐cell‐line correlation of Glut5 transport activity.

Quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors.

The importance of DNA polymerases in biology and biotechnology, and their recognition as potential therapeutic targets, drives development of methods for deriving kinetic characteristics of polymerases and their propensity to perform polynucleotide synthesis over modified DNA templates. Among various polymerases, translesion synthesis (TLS) polymerases enable cells to avoid the cytotoxic stalling of replicative DNA polymerases at chemotherapy-induced DNA lesions, thereby leading to drug resistance. Identification of TLS inhibitors to overcome drug-resistance necessitates the development of appropriate high-throughput assays. Since polymerase-mediated DNA synthesis involves the release of inorganic pyrophosphate (PPi), we established a universal and fast method for monitoring the progress of DNA polymerases based on the quantification of PPi with a fluorescence-based assay that we coupled to in vitro primer extension reactions. The established assay has a nanomolar detection limit in PPi and enables the evaluation of single nucleotide incorporation and DNA synthesis progression kinetics. The results demonstrated that the developed assay is a reliable method for monitoring TLS and identifying nucleoside and nucleotide-based TLS inhibitors.

Modulation of Cytotoxicity by Transcription-Coupled Nucleotide Excision Repair Is Independent of the Requirement for Bioactivation of Acylfulvene

Bioactivation as well as DNA repair affects the susceptibility of cancer cells to the action of DNA-alkylating chemotherapeutic drugs. However, information is limited with regard to the relative contributions of these processes to the biological outcome of metabolically activated DNA alkylating agents. We evaluated the influence of cellular bioactivation capacity and DNA repair on cytotoxicity of the DNA alkylating agent acylfulvene (AF). We compared the cytotoxicity and RNA synthesis inhibition by AF and its synthetic activated analogue iso-M0 in a panel of fibroblast cell lines with deficiencies in transcription-coupled (TC-NER) or global genome nucleotide excision repair (GG-NER). We related these data to the inherent bioactivation capacity of each cell type on the basis of mRNA levels. We demonstrated that specific inactivation of TC-NER by siRNA had the largest positive impact on AF activity in a cancer cell line. These findings establish that transcription-coupled DNA repair reduces cellular sensitivity to AF, independent of the requirement for bioactivation.

Near-infrared fluorescent probe for sensitive detection of Pb(II) ions in living cells

A new near-infrared fluorescent probe (NIR-PbP) for sensitive detection of Pb(II) ions in solution and living cells has been rationally designed and synthesized. The NIR-PbP is inherently non-fluorescent and gains fluorescence in the presence Pb(II) ions. The ion detection is based on Pb(II)-induced unmasking the fluorophore through the opening of the spyrocycle, with more than 500-fold fluorescence for sub-micromolar Pb(II) concentration. The NIR-PbP has high sensitivity, good photo-stability, low detection limit, and reversible response to Pb(II) ions.

Molecular Tools for Facilitative Carbohydrate Transporters (Gluts)

Facilitative carbohydrate transporters—Gluts—have received wide attention over decades due to their essential role in nutrient uptake and links with various metabolic disorders, including diabetes, obesity, and cancer. Endeavors directed towards understanding the mechanisms of Glut‐mediated nutrient uptake have resulted in a multidisciplinary research field spanning protein chemistry, chemical biology, organic synthesis, crystallography, and biomolecular modeling. Gluts became attractive targets for cancer research and medicinal chemistry, leading to the development of new approaches to cancer diagnostics and providing avenues for cancer‐targeting therapeutics. In this review, the current state of knowledge of the molecular interactions behind Glut‐mediated sugar uptake, Glut‐targeting probes, therapeutics, and inhibitors are discussed.

Fluorescent probes based on π-conjugation modulation between hemicyanine and coumarin moieties for ratiometric detection of pH changes in live cells with visible and near-infrared channels

We report two ratiometric fluorescent probes based on π-conjugation modulation between coumarin and hemicyanine moieties for sensitive ratiometric detection of pH alterations in live cells by monitoring visible and near-infrared fluorescence changes. In a π-conjugation modulation strategy, a coumarin dye was conjugated to a near-infrared hemicyanine dye via a vinyl connection while lysosome-targeting morpholine ligand and o-phenylenediamine residue were introduced to the hemicyanine dye to form closed spirolactam ring structures in probes A and B, respectively. The probes show only visible fluorescence of the coumarin moiety under physiological and basic conditions because the hemicyanine moieties retain their closed spirolactam ring structures. However, decrease of pH to acidic condition causes spirolactam ring opening, and significantly enhances π-conjugation within the probes, thus generating new near-infrared fluorescence peaks of the hemicyanine at 755 nm and 740 nm for probes A and B, respectively. Moreover, the probes display ratiometric fluorescence response to pH with decreases of the coumarin fluorescence and increases of the hemicyanine fluorescence when pH changes from 7.4 to 2.5. The probes are fully capable of imaging pH changes in live cells with good ratiometric responses in visible and near-infrared channels, and effectively avoid fluorescence blind spots under neutral and basic pH conditions - an issue that typical intensity-based pH fluorescent probes run into. The probe design platform reported herein can be easily applied to prepare a variety of ratiometric fluorescent probes for detection of biological thiols, metal ions, reactive oxygen and nitrogen species by introducing appropriate functional groups to hemicyanine moiety.

Metabolism-Driven High-Throughput Cancer Identification with GLUT5-Specific Molecular Probes

Point-of-care applications rely on biomedical sensors to enable rapid detection with high sensitivity and selectivity. Despite advances in sensor development, there are challenges in cancer diagnostics. Detection of biomarkers, cell receptors, circulating tumor cells, gene identification, and fluorescent tagging are time-consuming due to the sample preparation and response time involved. Here, we present a novel approach to target the enhanced metabolism in breast cancers for rapid detection using fluorescent imaging. Fluorescent analogs of fructose target the fructose-specific transporter GLUT5 in breast cancers and have limited to no response from normal cells. These analogs demonstrate a marked difference in adenocarcinoma and premalignant cells leading to a novel detection approach. The vastly different uptake kinetics of the analogs yields two unique signatures for each cell type. We used normal breast cells MCF10A, adenocarcinoma cells MCF7, and premalignant cells MCF10AneoT, with hepatocellular carcinoma cells HepG2 as the negative control. Our data indicated that MCF10AneoT and MCF7 cells had an observable difference in response to only one of the analogs. The response, observed as fluorescence intensity, leads to a two-point assessment of the cells in any sample. Since the treatment time is 10 min, there is potential for use in rapid on-site high-throughput diagnostics.

Altered Minor‐Groove Hydrogen Bonds in DNA Block Transcription Elongation by T7 RNA Polymerase

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base‐pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H‐bonding capacities. The results suggest that introducing wobble base‐pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base‐pairs do not H‐bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base‐pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription‐coupled DNA repair.

Targeting Sugar Uptake and Metabolism for Cancer Identification and Therapy: An Overview

Metabolic deregulations have emerged as a cancer characteristic, opening a broad avenue for strategies and tools to target cancer through sugar uptake and metabolism. High expression levels of sugar transporters in cancer cells offered glycoconjugation as an approach to achieve enhanced cellular accumulation of drugs and imaging agents, with the sugar moiety anchoring the bioactive cargo to cancer cells. On the other hand, high demand for sugar nutrients in cancers provided a new avenue to target cancer cells with metabolic or sugar uptake inhibitors to induce cancer cells starvation or death. This overview summarizes recent advances in targeting cancer cells through sugar transport for cancer detection and therapy.