Search Results (58)

RelA/SpoT homologue family enzymes participate in controlling the cellular levels of the alarmone (p)ppGpp, thereby activating the stringent response and promoting survival under stress conditions. These proteins contain an N-terminal catalytic domain and a C-terminal regulatory domain. They catalyze both the synthesis of ppGpp/pppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate. Here, we report the crystal structure of the N-terminal domain of Rel from Streptococcus equisimilis in complex with pppGpp at 3.2 Å resolution. The asymmetric unit contains a dimer with asymmetric ligation: pppGpp occupies only the synthetase site in one monomer, whereas in the other monomer, it is bound in both the hydrolase and synthetase sites. The two monomers exhibit distinct conformational states, with pronounced rearrangements of the flexible loops surrounding the binding pockets, including the α2/α3 and α8/α9 loops that act as steric gates. Molecular dynamics simulations support the dual binding arrangement and reveal additional probable transient binding sites, including a region in the linker between hydrolase and synthetase subdomains. These findings provide a structural framework for understanding how pppGpp binding modulates the opposing catalytic activities of bifunctional Rel enzymes and suggest possible mechanisms for (p)ppGpp-mediated autoregulation. Full article

A convergent scheme for the preparation of conjugates with the dispiroindolinone-pyrrolidine-thioimidazolone and glutarimide moieties connected via a triazole-containing linker is proposed. Target conjugates were synthesized by azide–alkyne (3+2) cycloaddition reactions between propargylthio-substituted dispiroindolinone-pyrrolidine-imidazolones and an azido-glutarimide derivative. The starting compounds were available isothiocyanates, glycine, substituted benzaldehydes, chloroacetamide, and ethyl acrylate. The key azide–alkyne (3+2) cycloaddition step was carried out using TBTA as a catalyst, achieving >70% product yields. The resulting bifunctional compounds contained a fragment of dispiroindolinone (a p53-MDM2 interaction inhibitor) and glutarimide, a ubiquitin ligase ligand. The obtained dispiroindolinone-glutarimide conjugates were tested for their potential as hetero-PROTAC compounds for p53 reactivation. Individual conjugates showed preferential cytotoxicity against HCT116 colon carcinoma cells (wild-type53) compared to the isogenic HCT116p53^−/− subline. Full article

The oncogenic transcription factor MYC is a key driver of the development and progression of various types of cancer, but its intrinsically disordered structure and dependence on protein–protein interactions make it a difficult therapeutic target. Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules that can induce the selective degradation of disease-relevant proteins. In this study, we report the synthesis and biological testing of a series of novel MYC-targeted PROTACs derived from the MYC inhibitor EN4. These ligands were conjugated to cereblon (CRBN) or von Hippel–Lindau (VHL) E3 ligase recruiters using different linker architectures and connection sites. The resulting PROTACs were synthesized in high purity and characterized analytically. Cellular evaluation in HEK293T, Panc-1 and HCT-116 cancer cells revealed only moderate reductions in cell viability. Unfortunately, none of the synthesized PROTACs showed detectable MYC degradation at biologically relevant concentrations. Testing the stability of the PROTACs in microsomes showed rapid degradation, which may be a reason for the observed inactivity in cells. These results underscore the significant challenges associated with the targeted protein degradation of intrinsically disordered transcription factors such as MYC. Further studies are necessary to identify additional causes for the lack of MYC degradation and to optimize the chemical structures accordingly. Full article

Polymyxin antibiotics are often the last line of defense against multidrug-resistant Gram-negative pathogens. A key resistance mechanism involves the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, mediated by the bifunctional enzyme ArnA. However, the evolutionary rationale and structural basis for ArnA’s domain fusion, hexameric assembly, and catalytic coordination remain mechanistically unresolved. Here, we integrate evolutionary genomics, high-resolution cryo-electron microscopy (cryo-EM), and computational protein design to provide a comprehensive mechanistic analysis of ArnA. Our evolutionary analysis reveals that the dehydrogenase (DH) and formyltransferase (TF) domains evolved independently and were selectively fused in Gammaproteobacteria, suggesting an adaptive advantage. A 2.89 Å cryo-EM structure of apo-ArnA resolves the flexible interdomain linker and reveals a DH-driven hexameric architecture essential for enzymatic activity. 3D variability analysis captures intrinsic conformational dynamics, indicating a molecular switch that may coordinate sequential catalysis and substrate channeling. Structure-based peptide inhibitors targeting the hexamerization and predicted ArnA–ArnB interaction interfaces were computationally designed, offering a novel strategy for disrupting L-Ara4N biosynthesis. These findings illuminate a previously uncharacterized structural mechanism of antimicrobial resistance and lay the groundwork for therapeutic intervention. Full article

Clove oil is an essential oil used in food and pharmaceutical applications, with a market value of 300+ million dollars per year. Microemulsions have been used as effective clove oil delivery vehicles and could also be used to develop new extraction processes from clove buds. Eugenol, the main component of clove oil, is a polar oil that behaves as a surfactant and as an oil. This bifunctional behavior makes formulating clove oil microemulsions a challenging task. Here, we used a version of the Hydrophilic–Lipophilic Difference (HLD) + Net-Average Curvature (NAC) model that incorporates the bifunctional polar oil model to predict and fit the phase behavior of lecithin (surfactant) + polyglycerol-10 caprylate (hydrophilic linker) microemulsions using mixtures of heptane and clove oil as the oil phase. Using HLD-NAC parameters from the literature, the predicted HLD-NAC curves reproduced the expected phase transitions and the trends in Eugenol segregation toward the surfactant layer. Using these literature parameters as an initial guess to fit the experimental phase volumes produced accurate calculated phase volumes, and predicted interfacial tensions. This work demonstrates the application of heuristics and databases of HLD-NAC parameters in predicting the complex phase behavior of surfactant–oil–water (SOW) systems. Full article

The advancement of photocatalytic materials is critical for addressing environmental challenges such as water remediation, where efficient, robust, and reusable systems are in high demand. In this search, the development of hierarchically organized photocatalytic configurations with spatial control over active sites can significantly enhance performance. With this in mind, we present here a novel biomimetic approach for the patterned growth of TiO₂-ZnO photocatalytic heterostructures using solid-binding peptides (SBPs) as molecular linkers. Specifically, using bi-functional SBPs with selective affinity for both oxides, we achieve site-specific, molecularly guided deposition of TiO₂ nanoparticles onto pre-patterned ZnO-coated substrates. Leveraging the specific recognition capabilities and strong binding affinities of the engineered SBPs, the proposed biomimetic methodology allows for the fabrication of well-organized hybrid nanostructures under sustainable conditions. Photocatalytic degradation assays employing methyl orange as a model contaminant indicate that the patterned architecture enhances both the accessibility of the active photocatalytic sites and the recoverability of the material. This reusability is a critical parameter for the practical deployment of photocatalytic systems in water purification technologies. The obtained results underscore the potential of SBP-mediated molecular recognition as a versatile tool for green nanofabrication of functional materials with advanced architectural and catalytic properties. Full article

SARS-CoV-2 entry into host cells involves multiple steps and is a highly orchestrated process. Both the host protease TMPRSS2 and the HR1/HR2 segment within the spike (S) protein play a crucial role in promoting viral invasion. Herein, we report a series of bifunctional SARS-CoV-2 entry inhibitors formed by covalently linking a TMPRSS2 inhibitor, Camostat (Cm), and an HR1-targeting peptide fusion inhibitor IPB19 via a poly (ethylene glycol) (PEG) linker. Among them, IP4X and IP4Z display potent inhibitory activities against SARS-CoV-2 with similar IC₅₀ values of 0.16 μM and 0.17 μM, respectively. The efficacy surpassed that of their parent inhibitors by approximately 28-fold relative to Camostat and 15-fold relative to IPB19. We confirm that IP4X and IP4Z exhibit a dual-targeting mechanism by binding to both TMPRSS2 and HR1 region of S protein. These findings highlight the potential of the bifunctional inhibitors for further development as a novel multitarget therapy against SARS-CoV-2 infection and enrich the understanding of S-mediated entry of SARS-CoV-2 into host cells. Full article

Industrial pollution caused by Cu(II) ions remains one of the most critical environmental challenges worldwide. A novel green-fluorescent aerogel has been successfully developed for simultaneous sensing and adsorption of Cu(II) through the cross-linking of carboxymethyl nanocellulose and carbon dots (C dots) using epichlorohydrin as a linker. The C dots were synthesized by heating glucose and aspartate mixed solutions at 150 °C. Under UV illumination, the aerogel exhibited intense homogeneous green fluorescence originating from the uniformly dispersed C dots, whose emission can be efficiently quenched by Cu(II) ions. By leveraging smartphone-based imaging, the concentration of Cu(II) was quantified within the range of 5–200 µg/L, with a detection limit of 3.7 µg/L. The adsorption isotherm of Cu(II) onto the aerogel strictly conformed to the Freundlich isotherm model (fitting coefficient R² = 0.9992), indicating a hybrid adsorption mechanism involving both physical adsorption and chemical complexation. The maximum adsorption capacity reached 149.62 mg/g, a value surpassing many reported adsorbents. X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy analyses confirmed that the interactions between the aerogel and Cu(II) involved chelation and redox reactions, mediated by functional groups such as hydroxyl, amino, and carboxyl moieties. The straightforward fabrication process of the aerogel, coupled with its low cost, abundant raw materials, facile synthesis, and superior Cu(II) removal efficiency, positions this bifunctional fluorescent material as a promising candidate for large-scale environmental remediation applications. Full article

The removal of pharmaceutical contaminants like the anticonvulsant carbamazepine (CBZ) from water sources is a growing environmental challenge. This study explores the development of molecularly imprinted polymers (MIPs) tailored for CBZ adsorption using a bulk polymerization approach. Initially, this study focused on selecting the optimal cross-linker, comparing a trifunctional (trimethylolpropane triacrylate, TRIM) and a bifunctional cross-linker (ethylene glycol dimethacrylate, EGDMA) in combination with two common monomers (2-vinylpyridine and methacrylic acid). TRIM-based MIPs demonstrated superior adsorption efficiency and stability due to their higher cross-linking density. To improve sustainability, six bio-based monomers were investigated; of these, eugenol (EUG) and coumaric acid (COU) showed the best CBZ affinity due to π-π interactions and hydrogen bonding. Adsorption tests conducted in pharmaceutical-spiked real wastewater demonstrated that MIPs exhibit a high selectivity for CBZ over other pharmaceuticals like the anti-inflammatory drugs diclofenac (DCF) and ibuprofen (IBU), even at high concentrations. Reaction conditions were further optimized by adjusting the reaction time and the ratio between reagents to enhance selectivity and adsorption performance. These results highlight the potential of bio-based MIPs as efficient and selective materials for the removal of pharmaceutical pollutants from wastewater. Full article

Background: Fibroblast activation protein (FAP)-targeted theranostic radiopharmaceuticals have shown desired tumor-to-background organ selectivity due to the ubiquitous presence of FAP within the tumor microenvironment. However, suboptimal tumor retention and fast clearance have hindered their use to deliver effective cancer therapies. With well-documented FAP-targeting moieties and linkers appending them to optimal chelators, the development of copper radiopharmaceuticals has attracted considerable interest, given the fact that an ideal theranostic pair of copper radionuclides (⁶⁴Cu: t_1/2 = 12.7 h; 17.4% β⁺; E_β⁺_max = 653 keV and ⁶⁷Cu: t_1/2 = 2.58 d; 100% β⁻; E_β⁻_max = 562 keV) are available. Herein, we report our design, synthesis, and comparative evaluation of monovalent and divalent FAP-targeted theranostic conjugates constructed from our previously reported bifunctional chelator scaffold (BFS) based on 1,4,8,11-tetraaza-bicyclo [6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), which forms the most stable complex with Cu(II). Methods: After synthesis and characterization, the monovalent and divalent conjugates were radiolabeled with ⁶⁴Cu for in vitro cell assays, followed by in vivo positron emission tomography (PET) imaging evaluation in relevant mouse models. Results: Both ⁶⁴Cu-labeled conjugates showed high in vitro stability and anticipated FAP-mediated cell binding and internalization. The divalent one showed significantly higher FAP-specific tumor uptake than its monovalent counterpart. Conclusions: Our results demonstrate that the BFS-based multivalent approach can be practically used to generate FAP-targeted radiotheranostic agents for effective cancer diagnosis and treatment. Full article

Inhalable formulations with cyclodextrins (CDs) as solubility and absorption enhancers show promise for pulmonary delivery. Thiolated hydroxypropyl-β-cyclodextrin (HP-β-CD-SH) has mucoadhesive properties, enhancing drug absorption. Moreover, it has self-aggregation capability, which could further improve absorption and drug stability, as well as reduce irritation. This study aims to stabilize CD nanoaggregates using bifunctional cross-linkers and evaluate their benefits for lung drug delivery compared to pristine HP-β-CD-SH. Methods: The effectiveness of cross-linked HP-β-CD-SH nanoparticles (HP-β-CD-SH-NP) was compared to transient nanoaggregates in enhancing the activity of dexamethasone (DMS) and olive leaf extracts (OLE). DMS, a poorly soluble drug commonly used in lung treatments, and OLE, known for its antioxidant properties, were chosen. Drug-loaded HP-β-CD-SH-NP were prepared and nebulized onto a lung epithelial Air–Liquid Interface (ALI) model, assessing drug permeation and activity. Results: HP-β-CD-SH with 25% thiolation was synthesized via microwave reaction, forming 150 nm nanoaggregates and stabilized 400 nm HP-β-CD-SH-NP. All carriers showed good complexing ability with DMS and OLE and were biocompatible in the lung ALI model. HP-β-CD-SH promoted DMS absorption, while stabilized HP-β-CD-SH-NP protected against oxidative stress. Conclusion: HP-β-CD-SH is promising for lung delivery, especially as stabilized nanoaggregates, offering versatile administration for labile molecules like natural extracts. Full article

Human carbonic anhydrase IX (hCA IX) is a zinc(II)-dependent metalloenzyme that plays a critical role in the conversion of carbon dioxide and water to protons and bicarbonate. It is a membrane-bound protein with an extracellular catalytic center that is predominantly overexpressed in solid hypoxic tumors. Sulfamates and sulfonamides, for example acetazolamide (AZA), have been used to inhibit hCA IX in order to improve the response to solid hypoxic tumors. In the present study, we propose a new drug targeting approach by attaching the natural cytotoxic substances betulin and betulinic acid (BA) via a linker to sulfonamides. The conjugate was designed with different spacer lengths to accumulate at the target site of hCA IX. Computational and cell biological studies suggest that the length of the linker may influence hCA IX inhibition. Cytotoxicity tests of the newly synthesized bifunctional conjugates 3, 5, and 9 show effective cytotoxicity in the range of 6.4 and 30.1 µM in 2D and 3D tumor models. The hCA IX inhibition constants of this conjugates, measured using an in vitro enzyme assay with p-nitrophenyl acetate, were determined in a low µM-range, and all compounds reveal a significant inhibition of hypoxia-induced CA activity in a cell-based assay using the Wilbur–Anderson method. In addition, the cells respond with G1 increase and apoptosis induction. Overall, the dual strategy to produce cytotoxic tumor therapeutics that inhibit tumor-associated hCA IX was successfully implemented. Full article

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin–peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to β-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria. Full article

Overcoming the blood–brain barrier (BBB) remains a significant challenge with regard to drug delivery to the brain. By incorporating targeting ligands, and by carefully adjusting particle sizes, nanocarriers can be customized to improve drug delivery. Among these targeting ligands, transferrin stands out due to the high expression level of its receptor (i.e., transferrin receptor) on the BBB. Porous silicon nanoparticles (pSiNPs) are a promising drug nanocarrier to the brain due to their biodegradability, biocompatibility, and exceptional drug-loading capacity. However, an in-depth understanding of the optimal nanoparticle size and transferrin surface density, in order to maximize BBB penetration, is still lacking. To address this gap, a diverse library of pSiNPs was synthesized using bifunctional poly(ethylene glycol) linkers with methoxy or/and carboxyl terminal groups. These variations allowed us to explore different transferrin surface densities in addition to particle sizes. The effects of these parameters on the cellular association, uptake, and transcytosis in immortalized human brain microvascular endothelial cells (hCMEC/D3) were investigated using multiple in vitro systems of increasing degrees of complexity. These systems included the following: a 2D cell culture, a static Transwell model, and a dynamic BBB-on-a-chip model. Our results revealed the significant impact of both the ligand surface density and size of pSiNPs on their ability to penetrate the BBB, wherein intermediate-level transferrin densities and smaller pSiNPs exhibited the highest BBB transportation efficiency in vitro. Moreover, notable discrepancies emerged between the tested in vitro assays, further emphasizing the necessity of using more physiologically relevant assays, such as a microfluidic BBB-on-a-chip model, for nanocarrier testing and evaluation. Full article

The functionalization of inorganic surfaces by organic functional molecules is a viable and promising method towards the realization of novel classes of biosensing devices. The proper comprehension of the chemical properties of the interface, as well as of the number of active binding sites for bioreceptor molecules are characteristics that will determine the interaction of the sensor with the analyte, and thus its final efficiency. We present a new and reliable surface functionalization route based on supersonic molecular beam deposition (SuMBD) using 2,6-naphthalene dicarboxylic acid as a bi-functional molecular linker on the chemically inert silicon nitride surface to further allow for stable and homogeneous attachment of biomolecules. The kinetically activated binding of the molecular layer to silicon nitride and the growth as a function of deposition time was studied by X-ray photoelectron spectroscopy, and the properties of films with different thicknesses were investigated by optical and vibrational spectroscopies. After subsequent attachment of a biological probe, fluorescence analysis was used to estimate the molecular layer’s surface density. The successful functionalization of silicon nitride surface via SuMBD and the detailed growth and interface analysis paves the way for reliably attaching bioreceptor molecules onto the silicon nitride surface. Full article