ChemBioChem最新文献

The question of lanthanide (Ln) uptake in Ln-using bacteria has gained a lot of attention in recent years, and the existence of specific Ln-binding metallophores, termed lanthanophores, has been postulated. Here, the recently isolated metallophore methylolanthanin (MLL), which is shown to be involved in Ln metabolism of Methylobacterium extorquens AM1 along the structurally related siderophore rhodopetrobactin B (RPB B), is investigated. The total synthesis of both chelators as well as Ln-binding investigations employing a multitude of spectroscopic methods is reported. Compared to MLL, RPB B has a higher binding affinity for Fe³⁺. Unexpectedly, both metallophores seem to precipitate Lns under biologically relevant conditions (pH and concentration range). Therefore, a solubility product of -12.07 ± 0.24 mol² L^-2 for the precipitated Eu³⁺-MLL complex is reported. Furthermore, a combination of single-cell inductively coupled plasma mass spectrometry and Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of bacterial supernatant to investigate the Nd accumulation as well as MLL secretion under Fe limitation in M. extorquens AM1 is used. Finally, ion mobility spectrometry-mass spectrometry and quantum chemical calculations are used to investigate the RPB B and MLL complexation in the gas phase with Fe³⁺ and all rare earth elements (except Pm). The results challenge the classical siderophore-like Ln uptake (via simple solubilization) through MLL and underline again a potential complex interplay between Fe³⁺ and Ln³⁺ in microbial Ln uptake.

Human inflammatory caspases (caspase-1, -4, and -5) are key players in the innate immune response. These enzymes have been shown to cleave proinflammatory substrates, implicating them in many inflammatory disease states. Their activity is frequently assessed using in vitro fluorogenic assays, with all three human inflammatory caspases preferring the same WEHD tetrapeptide. The study examines the specificity of these enzymes C-terminal to the cleaved aspartate residue with Förster resonance energy transfer peptide-based assays using 7-methoxycoumaryl alanine [A(MCA)] as the donor and lysine-conjugated dabsyl [K(Dab)] as the quencher. The P₄-P₁ peptide sequences A(MCA)EHD, A(MCA)VAD, and A(MCA)QPD are varied on the C-terminal (prime) side of the peptide. Historically, caspase-4 and caspase-5 have been grouped together in their reactivity. Herein, caspase-5 only appreciably cleaves the A(MCA)EHDGK(Dab) peptide, whereas caspase-4 displays broader reactivity. All base sequences react more considerably with caspase-4 when a glycine is included C-terminal to Asp. The specificity of caspase-1 at this position varies based on the P₃-P₁ sequence of the peptide. These results highlight the interconnectedness of the prime and nonprime side amino acid sequences and the different behavior of each enzyme, which can be useful in understanding these potential drug targets.

The cover demonstrates the tumor spheroid formation by cancer cells when mixed with amyloid based KLMEI peptide sequence (sequence is in inset) hydrogels. The background shows the SEM image of the amyloid hydrogel fibrils mimicking the extra cellular matrix, which promotes growth and proliferation of MCF7 cells into 3D tumor spheroid. The illustration of a 24-well plate containing the drop cast 3D spheroids with a magnified confocal image of an actual MCF 7 spheroid exhibits live cells at the outer regions with a central necrotic core, reminiscent of in vivo tumors. More details can be found in the Research Article by Komal Patel, Samir K. Maji, and co-workers (DOI: 10.1002/cbic.202400595).

An approach combining virtual and nuclear magnetic resonance (NMR)-based screening is presented here to identify low molecular weight molecules (small molecules) targeting viral RNA elements from the SARS-CoV-2 genome. The so-called high-resolution RNA structural Fragment Assembly of RNA with Full-Atom Refinement 2 (FARFAR2) ensembles of the conserved 5^'-terminal stem-loop 1 (SL1) and the pseudoknot of the -1 programed frameshift element are used as targets for binding of small molecules of three different virtual libraries of compounds. The resulting hits predicted by virtual screening are probed for their binding to the two RNA elements by ligand- and target-based NMR experiments. The results demonstrate that the integration of virtual and experimental NMR screening efficiently identifies RNA-binding small molecules as start molecular entities to advance RNA-targeted antiviral therapies in an efficient manner. The strategy does not only apply to SARS-CoV-2, but also provides a rapid, highly specific route to discovering therapeutics for other RNA-based pathogens, highlighting the critical role of RNA structural data in enriching virtual drug discovery efforts.

Protein Z (PZ) is a vitamin-K-dependent glycoprotein that serves as an anticoagulant cofactor in blood. Although it is homologous to the serine proteases of blood coagulation, PZ has no enzymatic activity. It binds to the PZ-dependent protease inhibitor and supports inactivation of factor Xa. In addition, an inhibitory effect on thrombin is described. Clinically, changed PZ levels increase the risk for thrombosis. The related activated protein C (APC) is described as the first anticoagulant protein that binds heme under hemolytic conditions leading to its inhibition. However, the network of inhibitors of blood coagulation is still underexplored with respect to their role in heme-triggered effects and the regulation thereof. PZ seems to be an interesting candidate in this context due to its homology to APC. Using PZ-derived peptides as models for potential heme-binding sites in PZ together with in silico studies, one specific heme-binding site in PZ is identified. Binding studies on protein level demonstrate binding characteristics similar to APC. Finally, the inhibitory effect of PZ toward thrombin is increased in the presence of heme, providing also insights into a potential functional consequence of the PZ-heme interaction. In addition, the anticoagulant function of PZ is dampened in the presence of heme in an aPTT-based clotting assay, suggesting a tendency toward heme-induced prothrombotic actions. In future, this will support mapping the diverse effects of heme within blood coagulation.

The actin cytoskeleton plays a central role in cellular organization and dynamics, yet the tools available for targeting actin function remain limited. Cytochalasans represent a large class of actin-targeting natural products that are readily cell permeable with varying cytotoxicity and actin-targeting capabilities in mammalian cells. Their pharmacologic exploitation not only requires an understanding of their mode of action but also exact knowledge of how they can be derivatized without affecting their bioactivity. Herein, the design, synthesis, and evaluation of five fluorescently labeled [11]cytochalasan derivatives generated from pyrichalasin H (PyriH) and 19,20-epoxycytochalasin C (EpoxyCytoC) are reported. Guided by molecular docking, the C21-OAc moiety is identified as a promising site for tag attachment without disrupting actin binding. Semisynthetic modifications enable the conjugation of different dyes to PyriH and EpoxyCytoC scaffolds. Moreover, the effect of linkers separating cytochalasans and dyes is analyzed. Biological evaluation supplemented by in vitro assays to additionally interrogates their activities on the assembly of pure actin filaments revealing distinct activity profiles. Together, this study compares permeability, cytotoxicity, and actin binding of five novel [11]cytochalasan probes bearing substitutions of the C21-OAc moiety. These findings establish the C21-OAc position as a versatile functionalization site and provide a framework for developing next generation cytochalasan-based actin-targeting probes.

Protein phosphorylation is critical to selective gene expression; the proteins that regulate transcription are often phosphorylated at multiple sites. Serine and threonine phosphorylation in transcription factors such as NF-κB has been studied, and specific serines are involved in transcriptional activation. Tyrosine phosphorylation of NF-κB p50 subunit can also facilitate the NF-κB-mediated expression of CD40 protein. This study seeks to determine whether tyrosine phosphorylations at positions not normally phosphorylated in vivo could nonetheless affect protein expression mediated by NF-κB. The alterations studied included p50 analogs having pTyr at positions 59 and 61, which do not contain Tyr naturally, and an analog containing a metabolically stable tyrosine methylene phosphonate at position 60. Additionally, to explore the structural basis for enhanced NF-κB binding to CD40 promoter DNA by tyrosine phosphorylation, an analog of NF-κB p50 containing pTyr at both positions 60 and 82 is prepared. The results reflected changes in the ability of the modified NF-κBs containing an altered p50 subunit to bind to CD40 promoter DNA in vitro, and to direct the synthesis of CD40 in cellulo.

The ability to map protein-protein interactions (PPI) within living cells at high spatial resolution is essential for unraveling their roles in cellular biology. Although several super-resolution microscopy techniques are available, visualizing PPI below the diffraction limit of light across entire live cells remains a challenge. An approach is introduced that combines the chemogenetic split fluorescent reporter splitFAST2 with stimulated emission depletion (STED) microscopy for sub-diffraction imaging of PPI. As the fluorescence of splitFAST2 is activated only where the two proteins interact, this system allows for highly precise and unambiguous localization in live cells, enhanced further by the rapid super-resolution imaging capabilities of STED. The improved spatial resolution of the approach enabled us to precisely map the subcellular localization of inducible interactions or constitutive interactions. Beyond simply detecting PPIs below the diffraction limit in whole living cells, splitFAST2 also serves as the basis for fluorescent probes with very low background, enabling effective subdiffraction imaging of repetitive cellular structures like filamentous actin and microtubules.

Oligoarginine peptides are of interest as cell-penetrating peptides (CPPs) and play important roles in gene regulation and immune response. Complexation of oligoarginine peptides by the supramolecular host p-sulfonatocalix[4]arene (CX4) is well-established and has led to the use of CX4 derivatives as effective counterion activators for arginine-rich CPPs and to the application of CX4 in peptide sensing. Herein, a systematic binding study between oligoarginine peptides with CX4 is reported. The results show that the binding affinity increases with increasing peptide length from ≈10⁴M^-1 for the peptide H-Arg-Arg-OH to nanomolar affinities for peptides with more than four arginine residues. Within the series, C-terminal carboxamides show higher affinities than the respective carboxylates. In addition, the influence of fluorescent dye labeling is investigated with sulforhodamine B (SRB) and fluorescein (FL) dye labels. Efficient fluorescent quenching is observed after complexation of the labeled peptides by CX4, which prompted the exploration of the complexes as reporter pairs for chemosensing applications. The results suggest that the combination of fluorescently labeled oligoarginine peptides with CX4 affords a modular platform of host-dye reporter pairs for the detection of polycationic peptides with tailorable excitation and emission wavelengths and tailorable binding affinity down to the nanomolar affinity range.

Hiring decisions are some of the most important choices PIs make for their research groups. Whether it is undergraduates, graduate students, postdoctoral researchers, or other research assistants, trainees are pivotal for the success of a research group. Choosing an effective team can lead to synergy amongst researchers, high creativity, and groundbreaking science. Choosing an ineffective team can lead to infighting, toxicity, low morale, and scientific stagnation. These same hiring decisions are critical in creating an equitable and inclusive lab environment. Importantly, the challenges for creating productive and inclusive lab groups align: both can be achieved by 1) advertising broadly to ensure a high number of qualified potential researchers apply and 2) removing unconscious selection bias in the hiring process such that these highly qualified researchers are hired. Intentionally and deliberately incorporating these steps into the recruiting and selection processes aids in the recruitment of a more effective and inclusive research team. Herein, practical advice for recruiting for academic research labs are described. We focus on three areas: advertising your research positions, assessing candidates for research positions, and assessing your hiring practices. In each, we give guiding principles and practical tips for designing an equitable and inclusive hiring process .