Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC₅₀=0.29 μM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.     

Leakage of Metallic Ball Seat Valves with Anisotropic Surfaces

Metallic ball seat valves are an essential component of many hydraulic systems. They are used for many different purposes such as pressure relief valves or check valves. Despite their universal usage, their sealing mechanism is not yet fully understood. In previous works, a successful method for the simulation of the fluid leakage of metallic ball seat valves has been developed and confirmed experimentally. The method is based on Persson's contact mechanics theory, which is based on surface roughness power spectrum C(q). This theory takes a wide range of roughness values at different length scales into account. The previous method has been restricted to isotropic surfaces, but most surfaces of practical interest are highly anisotropic. A method for the calculation of pressure flow factors using Persson's method is presented. Based on these, a model for the leakage calculation can be developed. The simulation results are validated using an experiment.     

Synthesis of Red-Light-Responsive Pheophorbide-a Tryptamine Conjugated Photosensitizers for Photodynamic Therapy

Six methyl pheophorbide-a derivatives were prepared by linking a tryptamine side chain at the C-13 ¹ , C-15 ² and C-17 ³ positions of pheophorbide-a. P repared conjugates were characterized and evaluated for their photocytotoxicity against A549 cells. The conjugate 6 a with strong absorption at 413 nm (Soret band), 663–671 nm (Q bands) and comparable fluorescence quantum yield (0.26) was found to exhibit significant cytotoxicity (659 nM). Molecular integration of pheophorbide-a and tryptamines showed synergistic effects as the most potent conjugate 6 a was identified with enhanced photocytotoxicity when compared to methyl pheophorbide-a. T he conjugate 6 a was smoothly taken up by A549 cells and exhibited intracellular localization predominantly to lysosome in the cytoplasm. Upon photoirradiation 6 a generated singlet oxygen to show potent cytotoxicity toward A549 cells.     

Innovative Diagnostic Peptide-Based Technologies for Cancer Diagnosis: Focus on EGFR-Targeting Peptides

Active targeting using biological ligands has emerged as a novel strategy for the targeted delivery of diagnostic agents to tumor cells. Conjugating functional targeting moieties with diagnostic probes can increase their accumulation in tumor cells and tissues, enhancing signal detection and, thus, the sensitivity of diagnosis. Due to their small size, ease of chemical synthesis and site-specific modification, high tissue penetration, low immunogenicity, rapid blood clearance, low cost, and biosafety, peptides offer several advantages over antibodies and proteins in diagnostic applications. Epidermal growth factor receptor (EGFR) is one of the most promising cancer biomarkers for actively targeting diagnostic and therapeutic agents to tumor cells due to its active involvement and overexpression in various cancers. Several peptides for EGFR-targeting have been identified in the last decades, which have been obtained by multiple means including derivation from natural proteins, phage display screening, positional scanning synthetic combinatorial library, and in silico screening. Many studies have used these peptides as a targeting moiety for diagnosing different cancers in vitro, in vivo, and in clinical trials. This review summarizes the progress of EGFR-targeting peptide-based assays in the molecular diagnosis of cancer.     

Imaging of KCa3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes

The Ca²⁺ activated K⁺ channel K_Ca3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this K_Ca3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [¹⁸F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of K_Ca3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms ( 9 a ) and with a F-atom and a methoxy moiety ( 9 b ) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single K_Ca3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of K_Ca3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the K_Ca3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.     

Synthesis and Structural Characterization of Macrocyclic Plasmin Inhibitors

Two series of macrocyclic plasmin inhibitors with a C-terminal benzylamine group were synthesized. The substitution of the N-terminal phenylsulfonyl group of a previously described inhibitor provided two analogues with sub-nanomolar inhibition constants. Both compounds possess a high selectivity against all other tested trypsin-like serine proteases. Furthermore, a new approach was used to selectively introduce asymmetric linker segments. Two of these compounds inhibit plasmin with K_i values close to 2 nM. For the first time, four crystal structures of these macrocyclic inhibitors could be determined in complex with a Ser195Ala microplasmin mutant. The macrocyclic core segment of the inhibitors binds to the open active site of plasmin without any steric hindrance. This binding mode is incompatible with other trypsin-like serine proteases containing a sterically demanding 99-hairpin loop. The crystal structures obtained experimentally explain the excellent selectivity of this inhibitor type as previously hypothesized.     

Inhibition Studies on Carbonic Anhydrase Isoforms I,II, IX,and XII with a Series of Sulfaguanidines

Two novel sulfaguanidine series, six N-(N,N′-dialkyl/dibenzyl-carbamimidoyl) benzenesulfonamide derivatives and nine N-(N-alkyl/benzyl-carbamimidoyl) benzenesulfonamide derivatives, were obtained by desulfidative amination of easily accessible dimethyl arylsulfonylcarbonimidodithioates under catalyst- and base-free conditions. The newly synthesized compounds were tested for the inhibition of four different isozymes of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). Both series reported here were inactive against the off-target isozymes hCA I and II (K_i>100 μM). Interestingly, all investigated compounds inhibited both target isozymes hCA IX and XII in the submicromolar to micromolar ranges in which K_i values spanned from 0.168 to 0.921 μM against hCA IX and from 0.335 to 1.451 μM against hCA XII. The results indicated that N-(N-alkyl/benzyl-carbamimidoyl) benzenesulfonamides were slightly more potent inhibitors than N-(N,N′-dialkyl/dibenzyl-carbamimidoyl) benzenesulfonamides. Among the evaluated compounds, N-n-octyl-substituted N-carbamimidoylbenzenesulfonamide showed the most significant activity with a K_i value of 0.168 μM against hCA IX, which was four-fold more selective toward this isozyme versus hCA XII. Again, another derivative from N-(N-alkyl/benzyl-carbamimidoyl) benzenesulfonamide series, N-p-methylbenzyl-substituted N-carbamimidoylbenzenesulfonamide, demonstrated superior inhibitory activity against hCA XII with a K_i value of 0.335 μM.     

Flame retardant and antibacterial properties of PLA/PHMG-P/APP composites

Poly(lactic acid) (PLA) has evolved into a commodity polymer with numerous applications. However, its high flammability limits its viability as a perfect alternative to petrochemical engineering plastics. In this study, PLA was modified using polyhexamethyleneguanidine phosphate (PHMG-P) and ammonium polyphosphate (APP). The flame retardant performance of PLA/PHMG-P/APP was investigated based on the limiting oxygen index (LOI), vertical burning test (UL-94), thermogravimetric analysis (TGA), cone calorimetry (CC), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and Raman Spectrometry. Qualitative and quantitative methods were used to determine the antibacterial properties of PLA composites. The LOI of PLA-10% (P:A = 1:4) was 31.7% and was rated V-0 in the UL-94 V-0 test. The antibacterial properties of the composites reflected the antibacterial effects of PLA-10% (P: A = 1:4) against Escherichia coli and Staphylococcus aureus, with the antibacterial rates reaching 93.41% and 93.26%, respectively. PHMG-P and APP had a synergistic flame-retardant effect and improved the flame retardancy of PLA while exhibiting excellent antibacterial properties.     

Fabrication of submicron Li-rich Li2(Ti,Zr)O3 solid solution ceramics with sluggish grain growth rate

Doping and forming solid solution is an effective approach to tailor densification and grain growth. In this study, submicron Li₂(Ti,Zr)O₃ solid solution ceramics was successfully fabricated by a modified solid-state route for the first time. The use of appropriate starting powders can greatly reduce the synthesis temperature, and the preparation of Li₂(Ti,Zr)O₃ with submicron grain size is possible. The substitution of Ti by Zr will inhibit the phase transition from cubic to monoclinic structure, as well as the grain growth and pore removal. By doping 10-at.% Zr, the grain size was significantly decreased from several μm to 400 nm at 900°C, which contributes to a high conductivity eight times that of pure Li₂TiO₃. Moreover, after being heated at 900°C for 10 days, the grain size of Li₂(Ti,Zr)O₃ only increases to 5 μm; however, the grains of Li₂TiO₃ grows up to 16 μm with abnormal grain growth. The compositions of Li₂(Ti,Zr)O₃ are high uniform with no element segregation, indicating the sluggish grain growth rate is caused by the slow diffusion of Zr rather than segregation-induced solute drag. By adding excess Li and two-step sintering, the Li-rich Li₂(Ti,Zr)O₃ ceramic pebbles with high crush load of 50–60 N and small grain size of 300–500 nm were successfully fabricated. This work demonstrates a simple method for the synthesis of Li₂(Ti,Zr)O₃, which makes this material more widely accessible for exploration and also help accelerate its engineering application to be an advanced solid breeder.