A Facile NMR Method for Pre-MRI Evaluation of Trigger-Responsive T1 Contrast Enhancement.

There is a growing interest in developing paramagnetic nanoparticles as responsive magnetic resonance imaging (MRI) contrast agents, which feature switchable T1 image contrast of water protons upon biochemical cues for better discerning diseases. However, performing an MRI is pragmatically limited by its cost and availability. Hence, a facile, routine method for measuring the T1 contrast is highly desired in early-stage development. This work presents a single-point inversion recovery (IR) nuclear magnetic resonance (NMR) method that can rapidly evaluate T1 contrast change by employing a single, optimized IR pulse sequence that minimizes water signal for "off-state" nanoparticles and allows for sensitively measuring the signal change with "switch-on" T1 contrast. Using peptide-induced liposomal gadopentetic acid (Gd3+ -DTPA) release and redox-sensitive manganese oxide (MnO2 ) nanoparticles as a demonstration of generality, this method successfully evaluates the T1 shortening of water protons caused by liposomal Gd3+ -DTPA release and Mn2+ formation from MnO2 reduction. Furthermore, the NMR measurement is highly correlated to T1 -weighted MRI scans, suggesting its feasibility to predict the MRI results at the same field strength. This NMR method can be a low-cost, time-saving alternative for pre-MRI evaluation for a diversity of responsive T1 contrast systems.

A Toolkit for Engineering Proteins in Living Cells: Peptide with a Tryptophan-Selective Ru-TAP Complex to Regioselectively Photolabel Specific Proteins.

Using a chemical approach to crosslink functionally versatile bioeffectors (such as peptides) to native proteins of interest (POI) directly inside a living cell is a useful toolbox for chemical biologists. However, this goal has not been reached due to unsatisfactory chemoselectivity, regioselectivity, and protein selectivity in protein labeling within living cells. Herein, we report the proof of concept of a cytocompatible and highly selective photolabeling strategy using a tryptophan-specific Ru-TAP complex as a photocrosslinker. Aside from the high selectivity, the photolabeling is blue light-driven by a photoinduced electron transfer (PeT) and allows the bioeffector to bear an additional UV-responsive unit. The two different photosensitivities are demonstrated by blue light-photocrosslinking a UV-sensitive peptide to POI. Our visible light photolabeling can generate photocaged proteins for subsequent activity manipulation by UV light. Cytoskeletal dynamics regulation is demonstrated in living cells via the unprecedented POI photomanipulation and proves that our methodology opens a new avenue to endogenous protein modification.

Liposomal PHD2 Inhibitors and the Enhanced Efficacy in Stabilizing HIF-1α.

Prolyl hydroxylase domain-containing protein 2 (PHD2) inhibition, which stabilizes hypoxia-inducible factor (HIF)-1α and thus triggers adaptation responses to hypoxia in cells, has become an important therapeutic target. Despite the proven high potency, small-molecule PHD2 inhibitors such as IOX2 may require a nanoformulation for favorable biodistribution to reduce off-target toxicity. A liposome formulation for improving the pharmacokinetics of an encapsulated drug while allowing a targeted delivery is a viable option. This study aimed to develop an efficient loading method that can encapsulate IOX2 and other PHD2 inhibitors with similar pharmacophore features in nanosized liposomes. Driven by a transmembrane calcium acetate gradient, a nearly 100% remote loading efficiency of IOX2 into liposomes was achieved with an optimized extraliposomal solution. The electron microscopy imaging revealed that IOX2 formed nanoprecipitates inside the liposome's interior compartments after loading. For drug efficacy, liposomal IOX2 outperformed the free drug in inducing the HIF-1α levels in cell experiments, especially when using a targeting ligand. This method also enabled two clinically used inhibitors-vadadustat and roxadustat-to be loaded into liposomes with a high encapsulation efficiency, indicating its generality to load other heterocyclic glycinamide PHD2 inhibitors. We believe that the liposome formulation of PHD2 inhibitors, particularly in conjunction with active targeting, would have therapeutic potential for treating more specifically localized disease lesions.

Immune cell shuttle for precise delivery of nanotherapeutics for heart disease and cancer.

The delivery of therapeutics through the circulatory system is one of the least arduous and less invasive interventions; however, this approach is hampered by low vascular density or permeability. In this study, by exploiting the ability of monocytes to actively penetrate into diseased sites, we designed aptamer-based lipid nanovectors that actively bind onto the surface of monocytes and are released upon reaching the diseased sites. Our method was thoroughly assessed through treating two of the top causes of death in the world, cardiac ischemia-reperfusion injury and pancreatic ductal adenocarcinoma with or without liver metastasis, and showed a significant increase in survival and healing with no toxicity to the liver and kidneys in either case, indicating the success and ubiquity of our platform. We believe that this system provides a new therapeutic method, which can potentially be adapted to treat a myriad of diseases that involve monocyte recruitment in their pathophysiology.

Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation.

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.

Induction of apoptosis by Uncaria tomentosa through reactive oxygen species production, cytochrome c release, and caspases activation in human leukemia cells.

Uncaria tomentosa (Wild.) DC., found in the Amazon rain forest in South-America and known commonly as cat's claw, has been used in traditional medicine to prevent and treat inflammation and cancer. Recently, it has been found to possess potent anti-inflammation activities. In this study, we extracted cat's claw using four different solvents of different polarities and compared their relative influence on proliferation in human premyelocytic leukemia HL-60 cell lines. Cat's claw n-hexane extracts (CC-H), ethyl acetate extracts (CC-EA) and n-butanol extracts (CC-B) had a greater anti-cancer effect on HL-60 cells than those extracted with methanol (CC-M). Furthermore, CC-EA induced DNA fragmentation in HL-60 cells in a clearly more a concentration- and time-dependent manner than the other extracts. CC-EA-induced cell death was characterized by cell body shrinkage and chromatin condensation. Further investigating the molecular mechanism behind CC-EA-induced apoptosis, sells treated with CC-EA underwent a rapid loss of mitochondrial transmembrane (DeltaPsi(m)) potential, stimulation of phosphatidylserine flip-flop, release of mitochondrial cytochrome c into cytosol, induction of caspase-3 activity in a time-dependent manner, and induced the cleavage of DNA fragmentation factor (DFF-45) and PARP poly-(ADP-ribose) polymerase (PARP). CC-EA promoted the up-regulation of Fas before the processing and activation of procaspase-8 and cleavage of Bid. In addition, the apoptosis induced by CC-EA was accompanied by up-regulation of Bax, down-regulation of Bcl-X(L) and cleavage of Mcl-1, suggesting that CC-EA may have some compounds that have anti-cancer activities and that further studies using cat's claw extracts need to be pursued. Taken together, the results of our studies show clearly that CC-EA's induction of apoptosis in HL-60 cells may make it very important in the development of medicine that can trigger chemopreventive actions in the body.