Targeting the Dark Lipid Kinase PIP4K2C with a Potent and Selective Binder and Degrader

Phosphatidylinositol 5-phosphate 4-kinase, type II, gamma (PIP4K2C) remains a poorly understood lipid kinase with minimal enzymatic activity but potential scaffolding roles in immune modulation and autophagy-dependent catabolism. Achieving potent and selective agents for PIP4K2C while sparing other lipid and non-lipid kinases has been challenging. Here, we report the discovery of the highly potent PIP4K2C binder TMX-4102, which shows exclusive binding selectivity for PIP4K2C. Furthermore, we elaborated the PIP4K2C binder into TMX-4153, a bivalent degrader capable of rapidly and selectively degrading endogenous PIP4K2C. Collectively, our work demonstrates that PIP4K2C is a tractable and degradable target, and that TMX-4102 and TMX-4153 are useful leads to further interrogate the biological roles and therapeutic potential of PIP4K2C.     

Coherent electron scattering captured by an attosecond quantum stroboscope

We demonstrate a quantum stroboscope based on a sequence of identical attosecond pulses that are used to release electrons into a strong infrared (IR) laser field exactly once per laser cycle. The resulting electron momentum distributions are recorded as a function of time delay between the IR laser and the attosecond pulse train using a velocity map imaging spectrometer. Because our train of attosecond pulses creates a train of identical electron wave packets, a single ionization event can be studied stroboscopically. This technique has enabled us to image the coherent electron scattering that takes place when the IR field is sufficiently strong to reverse the initial direction of the electron motion causing it to rescatter from its parent ion.     

Biochemometry-Based Discovery of Phenylpropanoids from Azadirachta indica Fruits as Inhibitors of In Vitro Osteoclast Formation

(1) Background: Inhibition of osteoclast differentiation is the key approach in treating osteoporosis. However, using state-of-the-art treatments such as bisphosphonates and estrogen-based therapy is usually accompanied by many side effects. As opposed to this, the use of natural products as an osteoporotic remedy delivers promising outcomes with minimal side effects. (2) Methods: In the present study, we implemented a biochemometric workflow comprising (i) chemometric approaches using NMR and mass spectrometry and (ii) cell biological approaches using an osteoclast cytochemical marker (TRAP). The workflow serves as a screening tool to pursue potential in vitro osteoclast inhibitors. (3) Results: The workflow allowed for the selective isolation of two phenylpropanoids (coniferyl alcohol and sinapyl alcohol) from the fruits of neem tree (Azadirachta indica). These two isolated phenylpropanoids showed a very promising dose-dependent inhibition of osteoclast differentiation with negligible effects in terms of cell viability. (4) Conclusion: The presented workflow is an effective tool in the discovery of potential candidates for osteoclast inhibition from complex extracts. The used biochemometric approach saves time, effort and costs while delivering precise hints to selectively isolate bioactive constituents.     

Electrochemical behavior of methyl viologen at graphite electrodes modified with Nafion sol–gel composite

Electrodes were prepared by spin-coating spectroscopic graphite rods with a Nafion doped sol. Coating solutions consisting of Nafion:TEOS (tetraethoxysilane) ratios of 3:1 and 4:1 gave smooth films on the electrode surface. These modified electrodes were evaluated and compared with Nafion modified and bare spectroscopic graphite electrodes using methyl viologen (MV²⁺) as a representative cationic electroactive probe. Substantial partitioning of MV²⁺ into the Nafion:sol–gel matrix to the electrode surface was observed by cyclic voltammetry and square wave voltammetry. Cyclic voltammograms of MV²⁺ in 0.1 M NaCl at Nafion:sol–gel 4:1 modified electrodes showed a reversible reduction to MV⁺ with E^0′=−0.695 V vs. Ag/AgCl. Results of scan rate variation showed the wave to be characterized by semi-infinite diffusion for scan rates in the range 50–500 mV/s. Slowing the scan rate below 50 mV/s resulted in a transition to thin-layer behavior. MV²⁺ partitioned much more quickly into the sol–gel-Nafion modified electrodes compared to pure Nafion modified electrodes. Reversibility of the MV²⁺-loaded modified Nafion-doped sol–gel coatings on electrodes was obtained by soaking in 1 M NaCl solution. Concentration calibration plots for MV²⁺ at the sol–gel-Nafion modified electrodes were nonlinear. Substantial enhancement of current signal at low concentrations was observed by square wave voltammetry.     

Traceless solid-phase synthesis and biological evaluation of purine analogs as inhibitors of multidrug resistance protein 4

The traceless solid-phase syntheses of 6-oxopurines and pyrazolo[3,4-d]pyrimidines are presented. The effects of these compounds on multidrug resistance protein 4 (MRP4/ABCC4) facilitated efflux was examined. Four of the compounds, 7b, 7c, 15a, and 17e, were active in inhibiting MRP4-mediated efflux of the bimane-glutathione conjugate. In addition, all four compounds were also able to reverse MRP4-mediated resistance to the anticancer drug 6-thioguanine. In the presence of 25 microM 15a or 17e, there was complete reversal. The reversal of resistance was achieved without any effects on the uptake and metabolism of 6-thioguanine.     

Surface modification of cellulose with plant triglycerides for hydrophobicity

Hydrophobic cotton was achieved by surface modification of the cellulose with triglycerides from several plant oils including soybean, rapeseed, olive and coconut oils. These oils were delivered to the cellulose substrates in homogeneous solutions of ethanol or acetone as well as aqueous emulsions. Surface modification was facilitated by solvent evaporation followed by heating between 110 and 120 °C for 60 min. All oils, except for coconut, produced hydrophobic and less water-absorbing cotton, supporting the desirable role of higher unsaturation in the fatty acids to achieve crosslinked network. The most hydrophobic surfaces were obtained by the reaction with 1% soybean oil in acetone. On both bleached and scoured cotton, a water contact angle of 80° and water absorption value of 0.82 μL/mg were achieved. The acquired hydrophobicity was not only retained after water washing but also improved with subsequent exposures to elevated temperatures. The surface tension of scoured cotton cellulose was lowered from 63.81 mJ/m² to 25.74 mJ/m² when modified by soybean oil delivered in acetone, which is lower than that of poly(ethylene terephthalate). An aqueous emulsion of soybean oil also rendered the scoured cotton hydrophobic, which shows promise for a green chemistry and bio-based approach to achieve water repellency on cellulosic materials.     

Fluorinated Photodynamic Therapy Device Tips and their Resistance to Fouling for In Vivo Sensitizer Release

We describe progress on a one‐step photodynamic therapy (PDT) technique that is simple: device tip delivery of sensitizer, oxygen and light simultaneously. Control is essential for their delivery to target sites to generate singlet oxygen. One potential problem is the silica device tip may suffer from biomaterial fouling and the pace of sensitizer photorelease is slowed. Here, we have used biomaterial (e.g. proteins, cells, etc.) from SQ20B head and neck tumors and whole blood for an assessment of fouling of the silica tips by adsorption. It was shown that by exchanging the native silica tip for a fluorinated tip, a better nonstick property led to an increased sensitizer output by ~10%. The fluorinated tip gave a sigmoidal photorelease where singlet oxygen is stabilized to physical quenching, whereas the native silica tip with unprotected SiO–H groups gave a slower (pseudolinear) photorelease. A further benefit from fluorinated silica is that 15% less biomaterial adheres to its surface compared to native silica based on a bicinchoninic acid assay (BCA) and X‐ray photoelectron spectroscopy (XPS) measurements. We discuss how the fluorination of the device tip increases biofouling resistance and can contribute to a new pointsource PDT tool.     

Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.