ChemBioChem最新文献

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.

Given the extracellular matrix (ECM) essential role in tissue development, maintenance, and repair, exploiting its glycan structures offers new opportunities for dynamic drug storage and targeted delivery of peptides via injectable solutions. This study investigates the reversible, pH–driven interaction between boronic acid-functionalized compounds and diol-rich glycans within cell-derived decellularized extracellular matrices (CDMs). An N-terminally functionalized model peptide was produced using carboxy-phenyl-boronic acid (CPBA), and its pH-dependent binding to the diol-rich structures of CDMs was demonstrated. A CPBA-conjugated myostatin inhibitor maintained its full potency as a prerequisite for future therapeutic application. The findings suggest N-terminally boronic acid-modified peptides as drug candidates for targeted delivery and storage to diol-rich glycans of the natural ECM.

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 .

Aberrant aggregation of β-amyloid (Aβ) peptides into insoluble fibrils is recognized as one of the hallmarks of Alzheimer's disease (AD). Among the post-translational modifications influencing Aβ behavior, nitration and nitrosation by nitric oxide (NO) derivatives play a key role, though their impact on aggregation and toxicity remains unclear. This contribution explores the effects on Aβ aggregation induced by an NO photodonor (NOPD) releasing two NO molecules via a stepwise mechanism under the control of visible blue light. Significant reduction of protein aggregation is observed when a considerable amount NO (≈120 µM) is photoreleased during the early stages of the protein aggregation. In contrast, long-term release of a similar NO concentration is ineffective. On the other hand, proaggregative effect is induced by the NOPD kept in the dark. The stable photoproducts formed after the release of the first and the second molecule of NO also show a protein aggregation inhibitory effect both individually and in combination. Dynamic simulation studies are also reported to shed light on the binding of NOPD and its photoproducts with key Aβ_1–40 residues.