ACS Bio & Med Chem Au最新文献

Currently, inteins are some of the most popular multifunctional tools in the fields of molecular biology and biotechnology. In this study, we used the surface analysis method to identify the sites of intermolecular interactions between the N and C-parts of the Ssp DnaE intein. The obtained results were used to determine the key amino acids that define the binding energy and type of contact between intein subunits. In silico substitution of five neutral amino acids in the C-part of Ssp DnaE with methionine was validated by using oligomutagenesis of a previously assembled plasmid, which was then used for in vitro tests with HEK293 cells. GFP reconstruction assays were used to estimate changes in trans-splicing efficiency using quantitative metrics such as the number of GFP+ cells and median fluorescence intensity as well as qualitative metrics such as microphotography and fluorescence curve analysis using live-cell microscopy. The results of the in vitro tests revealed significantly decreased splicing efficiency in four out of six mutant variants, with no significant differences in the other two cases. Additionally, we performed metadynamics modeling to explain how these mutations affect the molecular mechanisms of intein-intein interactions. Finally, we found a positive correlation between the structural and free energy changes in the local minima distribution and the decrease in splicing efficiency in the I151M and A162M+A165M cases. The resulting method was used with control mutations that had an experimentally confirmed positive (A168H) or negative (T198A) effect on the splicing reaction. In summary, we propose a method of free energy surface analysis in collective variables for quick and visual evaluation of mutation effects. This approach could be applied for the development of new biotechnological and gene therapy products to overcome AAV capacity limitations.

Air pollution exposure is linked to diseases spanning multiple physiological systems. However, environmental stress is overwhelmingly associated with several lung diseases. Since the chemical composition of air pollutants varies widely across geographical locations, research on how specific components in pollutant mixtures contribute to cellular dysfunction is needed. In this work, we utilized microscopy-based morphological profiling as a tool to assess the cellular susceptibility to pollutants. Through our analysis, we established morphological profiles of formaldehyde-exposed cells that showed dose-dependent shifts in cell shape profiles correlating with overall cell health. As a real-world proof-of-concept validation, we evaluated the differences in particulate matter (PM) composition across multiple geographical areas, including both urban and suburban communities in Austin, Texas, USA. Data from this real-world study was used to inform a multicombinatorial study involving metals, selenium (Se) and manganese (Mn), which were differentially abundant across PM collection sites. As proof of concept to demonstrate the potential of establishing low-cost, high-throughput combinatorial screening of the biological effects of these metals, we incorporated microfluidic technology to simultaneously generate variable two-component metal mixtures in a multiwell plate format that enabled microscopy-based morphological screening as a proxy for toxicity. Combinatorial analysis of Se and Mn showed dynamic cell responses across a range of concentrations. Interestingly, exposure mixtures containing both Se and Mn yielded healthier cellular phenotypes relative to Se exposure as a single component. These results demonstrate the development of a high-throughput pipeline to detect dynamic biological responses to common air pollutants. Leveraging multiple technologies, we demonstrate the feasibility of a cost-effective approach that can serve as a starting point to inform focused screenings and studies of air pollutant mixtures that affect health outcomes.

We report here on a straightforward methodology to synthesize a new water-soluble fluorescent probe Tris-BODIPY-OH 1 that contains three pH-independent hydrophilic arms. This probe has been prepared by exploiting a synthetic strategy that includes as a key step the combination of a Cu-(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and a Sonogashira cross-coupling in a sequential one-pot approach. Tris-BODIPY-OH 1 provides a significant advancement in the field by expanding the BODIPY toolbox with a biocompatible water-soluble probe, which can be used to specifically label and assess the function of the endoplasmic reticulum.

Proteins frequently undergo large-scale conformational excursions in their native ensemble. Such structural transitions are particularly critical for enabling access to binding sites when they are buried in the protein interior. Here, we map the conformational landscape of AlbAS, a natural isoform of the transcription factor AlbA from the gut microbe Klebsiella oxytoca, which sequesters the antibiotic albicidin in a solvent-inaccessible binding tunnel. Combining equilibrium, time-resolved experiments, structural mass spectrometry and calorimetry with statistical modeling, we show that AlbAS displays large differences in local and global stability and dynamics, with ∼600-fold difference in unfolding rates across different parts of the structure. Several residues lining the ligand-binding pocket and the inter-sub-domain residues rapidly exchange protons with the solvent in hydrogen-deuterium exchange mass spectrometry experiments, indicative of anisotropic distributions of local stabilities, with the N-terminal subdomain being less stable. The AlbAS conformational landscape is thus quite rugged, encompassing numerous partially structured states in equilibrium, including partial unlocking of the N-terminal subdomain at a time-constant of 6 ms that exposes the binding sites to aid in albicidin binding.

Farmers urgently need novel technologies to ensure global food security for the rapidly expanding population, yet crop protection has seen little innovation in decades. Mounting regulatory pressures, pest resistance, and environmental concerns are driving demand for novel sustainable solutions. Beyond traditional small molecule active ingredients, very few alternative modalities (e.g., peptides, RNAi, biopesticides) have reached the market. Meanwhile, targeted protein degradation (TPD) has emerged as a breakthrough modality for human therapeutics, with numerous proteolysis-targeting chimeras (PROTACs) and molecular glues (MGs) advancing through clinical trials. Those compounds induce potent and selective degradation of protein targets via the ubiquitin-proteasome system (UPS). Recently, PROTACs have been shown to function in both insect cells and in whole insect organisms, marking a pivotal step toward their use as next-generation crop protection solutions. In this perspective, we showcase the rationale, key challenges, and potential breadth of applications of targeted protein degraders for agricultural purposes. The TPD technology is a promising and potentially disruptive alternative to traditional small molecule inhibitors in agriculture.

Malaria still stands out as one of the most devastating and prevalent diseases globally, where the rise of resistance to different antimalarial drugs in different regions has posed significant obstacles to global treatment and elimination. Consequently, there is a pressing need for the development of new antimalarial agents with novel modes of action. In this study, we report the identification and optimization of new indole-2-carboxamide derivatives where structural modifications have yielded new compounds 6x with enhanced potency (Pf3D7-IC50 ∼ 0.3 μM) and improved metabolic stability (hMics = 3 μL/min/mg), while also minimizing the human ether-a-go-go-related gene (hERG, IC₅₀ > 20 μM) channel activity and cytotoxic effect on hepatic cells (CC₅₀ > 30 μM). Mode-of-action investigations revealed that a representative compound from this series interfered with homeostasis of the parasite's digestive vacuole. However, cross-resistance was observed with resistant strains, which was linked to efflux pumps such as Plasmodium falciparum chloroquine resistance transporter (PfCRT). Despite this challenge, these indole-2-carboxamides provide versatile molecular templates for innovative medicinal chemistry to overcome cross-resistance while maintaining other attractive properties of this novel series.

The most common cystic fibrosis mutation is the F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR), which causes misfolding of the first of two nucleotide binding domains (NBD1/2), preventing Mg/ATP-dependent NBD dimerization for normal function. Although folding correctors elexacaftor/VX-445 and lumacaftor/VX-809 have been combined to correct the NBD1 misfolding, the exact correction pathway is still unknown. In this study, the constrained tertiary noncovalent interaction networks or the thermoring structures of dimerized NBD1 in hCFTR/E1371Q with or without F508del were analyzed to identify the weakest noncovalent bridge as the final post-translational tertiary folding of dimerized NBD1 in response to folding correctors. These computational analyses suggested that hCFTR primarily used cooperative folding between α- and β-subdomains in dimerized NBD1 as the last step upon binding of the potentiator ivacaftor/VX-770. However, the binding of folding correctors allosterically protected the α-subdomain from misfolding until subsequent core formation. This thermodynamic protective mechanism, unlike the chaperone-based one in cotranslational NBD1 folding, may restore posttranslational NBD1 folding for tight Mg/ATP-mediated NBD dimerization in the F508del mutation and also potentially apply to treating other cystic fibrosis patients with rare mutations.

Streptococcus agalactiae also known as Group B Streptococcus (GBS) is a Gram-positive, encapsulated, pathogenic bacterium. GBS causes severe perinatal infections that lead to chorioamnionitis, funisitis, premature rupture of membranes, preterm birth, maternal sepsis, neonatal sepsis, stillbirth, and maternal demise. Epidemiological data indicate that the nutrient selenium provides protection against infection and adverse disease outcomes and is a critical nutrient for development of a healthy pregnancy. We hypothesized that selenium could have antimicrobial activity against GBS. To test this, we employed a panel of colonizing and invasive GBS strains and evaluated the bacterial growth in response to selenium exposure. Our results indicate that selenium can inhibit GBS growth and adherence to gestational tissues and that colonizing strains are more sensitive to selenium than invasive strains. Together, these results indicate that selenium could be deployed as a cost-effective intervention to ameliorate the risk of GBS perinatal infections.

Lectins, proteins that reversibly bind specific glycan motifs, offer dual utility as molecular probes or inhibitors of virus-host interactions. This review explores the molecular interactions between lectins and viral envelope glycoproteins, emphasizing their applications as antiviral agents and diagnostic tools. Enveloped viruses, such as HIV, Influenza, Herpesviruses, and Coronaviruses, exhibit dense glycosylation on their surface proteins, forming a glycan shield rich in high-mannose and complex glycans crucial for viral processes and immune evasion. Lectins exploit these glycan shields by selectively targeting conserved glycosylation sites on key viral proteins like gp120 (HIV), hemagglutinin (Influenza), spike (SARS-CoV-2), and glycoprotein D (HSV), thereby interfering with viral entry. Potent inhibitory activity across diverse virus families has been demonstrated for natural lectins such as griffithsin (GRFT), cyanovirin (CV-N), and banana lectin (BanLec), with novel fungal and algal lectins continually expanding the list. Concurrently, lectin-based biosensors utilizing electrochemical, plasmonic, and microfluidic platforms, often enhanced by nanomaterials or aptamers, enable sensitive and specific detection of glycosylated viral targets. Despite challenges including potential immunogenicity and production scalability, ongoing bioengineering efforts aim to refine lectin specificity, reduce toxicity, and enhance overall functionality. These collective advances showcase the role of lectins as versatile molecular tools for the detection, inhibition, and mechanistic study of viral pathogens.

We report the microwave-assisted synthesis of a novel family of peptide-linked optical imaging probes incorporating the L-[7,13] bombesin fragment (denoted L-[7,13]-BBN) as a functional building block currently used for targeting the gastrin-releasing peptide receptor (GRPR) in cancer cells. Given the importance of chirality in probe design, we synthesized and evaluated both L- and D-amino acid-substituted naphthalenediimide (NDI), namely, the monopeptide (L-3) and corresponding bis-peptide (L-4) conjugates. These bioconjugates were characterized using NMR, fluorescence spectroscopy, including excitation-emission mapping, and mass spectrometry, confirming their spectroscopic tunability, water solubility, and ability to form supramolecular aggregates. Aggregation behavior was demonstrated by scanning electron microscopy (SEM) and Time-Correlated Single-Photon Counting (TCSPC) spectroscopy, while circular dichroism studies revealed a stereochemistry-driven self-assembly influenced by 4-iodophenylalanine modifications. Additionally, a new, desymmetrized NDI-based bioconjugate (L-6), which incorporates the L-[7,13]-BBN fragment and a functional BODIPY fluorescent label, was synthesized in a stepwise manner via the microwave-assisted methods developed hereby. Cytotoxicity assays showed that these are benign, nontoxic probes at the time of imaging experiments and up to 72 h observation. Cellular uptake and localization properties of all compounds were assessed using confocal laser-scanning microscopy correlated with multiphoton fluorescence lifetime imaging microscopy (MP FLIM). This imaging method provided insights into the distinct behaviors of mono- vs bis-substituted peptide conjugates in live PC-3 prostate cancer cells, known to overexpress GRPR, and in A431 cells, known to overexpress the epidermal growth factor receptor (EGFR). Notably, the L- and D-stereochemistries of the BBN-[7,13] fragment played a crucial role in modulating the uptake and subcellular localization of bioconjugates of type 3 and 4 in lysosomes while the presence of the BODIPY unit additionally directed the biolocalization of compound L-6 toward the endoplasmic reticulum of multiple cellular environments, including in living PC-3 and A431 cells. These findings are relevant for the design of new biologically active probes, including proteolysis-inactive, peptide conjugates for cancer biomarker detection and imaging.