ACS Omega

Metallic nanoparticles have emerged as novel therapeutic agents due to their distinctive physicochemical properties and broad-spectrum activity, with applications in antimicrobial therapy, drug delivery and bioremediation. Conventional methods for metallic nanoparticle synthesis often utilize toxic chemicals and energy intensive processes that are expensive. Green synthesis offers a sustainable and cost-effective alternative by using biomolecules from plants and microorganisms. In this study, gold (AuNPs), silver (AgNPs), and copper oxide (CuO NPs) nanoparticles were biosynthesized using leaf extracts of Aloe africana Mill., a South African medicinal plant rich in phytochemicals, and the magnetotactic bacterium Magnetospirillum magnetotacticum that naturally produces intracellular nanoparticles. GC-MS analysis revealed 13 known phytochemicals in the A. africana extract including esters, terpenoids, monoglycerides, and fatty acids which served as reducing and capping agents for nanoparticle synthesis. A. africana-derived AgNPs were spherical (11-30 nm) in shape, capped with dihydrosqualene, a known antibacterial compound; and was found to display activity against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Enterococcus faecalis and Staphylococcus aureus) bacteria. These AgNPs however exhibited cytotoxicity to HEK293 and HeLa cell lines. A. africana AuNPs (17-62 nm) displayed diverse morphologies and CuO NPs (55-115 nm) were irregular shaped, and both nanoparticles exhibited limited antibacterial activity and low cytotoxicity. M. magnetotacticum-derived AuNPs (12-21 nm) and AgNPs (51-126 nm) were spherical, with the CuO NPs (42-66 nm) having irregular shapes. Except for A. africana-derived AgNPs, all other metallic nanoparticles displayed poor antibacterial activity. These findings are novel and highlight a dual-function green synthesis platform where A. africana phytochemicals contribute to both nanoparticle synthesis and bioactivity, positioning A. africana AgNPs as promising antibacterial agents.

Biocompatible fluorosurfactants are essential for many droplet microfluidic workflows but are often obtained from commercial sources because published syntheses of perfluoropolyether (PFPE)-based surfactants typically require acid chloride intermediates and chemistry-oriented purification methods. These requirements can limit access for biology and clinical laboratories seeking low-cost or customizable surfactant systems. Here we describe a practical method for preparing functional PFPE-based fluorosurfactant materials by direct carbodiimide coupling of functionalized PFPE carboxylic acids(Krytox 157 FSH) to amine-containing head groups under laboratory-accessible conditions. Using this approach, we prepared a PFPE-polyethylene-glycol (PFPE-PEG) material from Jeffamine ED900 and a PFPE-Tris material from Tris base. Because these products were not fully structurally characterized, we present them as functional reaction products and evaluate them by use in biomicrofluidic workflows rather than by definitive compositional assignment. PFPE-Tris was useful for generating relatively uniform small droplets, whereas the PFPE-PEG preparation supported a broader range of biological applications. These materials were used in genomic library screening for {beta}-glucosidase activity, thermocycling-associated droplet workflows, and protein crystallization experiments. In addition, the PFPE-PEG preparation improved emulsion behavior in many protein crystallization screens that were unstable with a commercial droplet oil used in our laboratory. This method reduces the practical barrier to in-house fluorosurfactant preparation and allows biology-focused laboratories to explore head-group chemistry, oil composition, and operating conditions without complete reliance on commercial reagents. The results support this workflow as a useful entry point for biomicrofluidics laboratories, while also highlighting the need for careful interpretation of thermocycled droplet assays and for future analytical characterization of the resulting materials. Significance statementDroplet microfluidics relies on fluorosurfactants that are often costly and difficult to synthesize outside of chemistry-focused settings. We describe a simple, biology-laboratory-compatible approach for generating functional perfluoropolyether-based fluorosurfactant materials using direct carbodiimide coupling and straightforward cleanup. The resulting materials supported multiple biomicrofluidic workflows in our laboratory, including enzymatic screening and protein crystallization, and provide a practical route for groups seeking lower-cost and more customizable surfactant systems.

In siRNA-based applications, cellular delivery remains one of the main hurdles. Many formulations are tested for the same and peptides came up as one of the optimal options. The latter have various advantages like natural biological presence, high specificity, and natural metabolism etc. siRNA in conjugation with peptides have exhibited enhanced mRNA silencing. Peptides aid siRNAs in condensation to smaller volumes, enhance nuclease protection, increase half-life, promote cell specific binding as well as endosomal escape and release in cytosol. Despite its prime importance, no resource is available for the peptide-based delivery of siRNAs, therefore to fill the gap we developed PEPsRNA web server. It includes 2266 entries of 270 different kinds of peptides, 106 different types of siRNAs and shRNAs along with more than 80 conjugate molecules targeting 55 different genes, experimentally tested for the delivery of the siRNAs. To provide the detailed insights of the procedure, we have incorporated analysis of the peptides (e.g. secondary structure, amino acid composition, polarity, hydrophobicity etc.), siRNAs (e.g. secondary structures with minimum free energies etc.) and associated conjugate molecules (e.g. structure, SMILES, Inchl). We have derived these values using various other tools and resources to make the web server comprehensive. We further compared various physicochemical properties with the efficacy of the peptide based on the target gene silencing, but these properties do not shown any distinct conclusive relationship. The data is available for browsing, searching and downloading freely on the web server with URL: http://bioinfo.imtech.res.in/manojk/pepsirna. Highlights PEPsRNA is the first database of experimentally tested peptides for siRNA delivery It comprised of 2266 entries with 270 peptides and about 80 conjugate molecules Analysis of peptides, siRNAs and details of conjugate molecules are provided Browse, search and various tools are incorporated for data retrieval and usage

The carbohydrate-binding proteins (LecA and LecB) present within the extracellular polymeric substance (EPS) matrix of Pseudomonas aeruginosa play an essential role in maintaining the structural integrity of the biofilms through interactions with the EPS polysaccharides. Therefore, targeting the above lectins can turn out to be one of the promising strategies for disrupting P. aeruginosa biofilms. In the current study, we investigated the potency of antimicrobial peptides (AMPs) produced by the human gut microbiome in targeting LecA and LecB proteins of P. aeruginosa. Initially, a comprehensive in-silico pipeline was developed to identify and characterize putative antibacterial and antibiofilm AMPs produced by the human gut microbiome. These AMPs were then subsequently studied for their interaction with the lectin proteins through molecular docking, MM-GBSA, residue analysis, and molecular dynamics (MD) simulation. Among the studied peptides, amp21 and amp24 exhibited the strongest interactions with the lectin protein, occupying binding sites overlapping with key active-site residues previously reported for raffinose binding. amp6, amp21, and amp24 were selected for in vitro validation based on the MD simulation results of both LecA and LecB proteins. The above selected peptides exhibited minimal hemolytic activity across the tested concentration range. amp21 and amp6 were non-toxic to mammalian cells while amp24 demonstrated cytotoxicity only at higher doses. amp21 was found to be the most potent AMP and inhibited the growth of P. aeruginosa by [~]60% at 50 {micro}g mL{square}{superscript 1}. amp6 and amp21 resulted in a significant disruption of P. aeruginosa biofilms. Membrane permeabilization assays and scanning electron microscopy revealed that amp6, amp21, and amp24 damaged the bacterial cell membranes apart from compromising the integrity of the biofilm EPS matrix. Lastly, through in-silico studies, we designed ultrashort peptides (USPs) from the lead AMPs. The USPs (amp21.4 and amp24.2) exhibit superior antibiofilm efficacy compared to their parent AMPs. These findings establish human-gut microbiome-derived AMPs as promising candidates to target P. aeruginosa biofilms via inhibition of lectin proteins.

Polymeric 3D printing of microfluidic devices for biosensing is an appealing fabrication alternative for rapid manufacturing of biosensing devices with complex geometry in a streamlined, repeatable and cost-effective manner without the need for expensive instrumentation such as those employed in photochemical etching and soft lithography. Hybrid 3D printed paper-based microfluidics is an emerging area which harnesses the unique properties of both, merging the construction of microfluidic structures and the inherent capillary-driven flow within paper substrates. In this work, we have fabricated hydrophobic barriers by 3D printing a single layer of machinable wax, thermoplastic polyurethane, polylactic acid and polypropylene directly on chromatography paper to create open microchannels and determine the most suitable material. Characterization of each open microchannel using the four materials revealed polypropylene as the most reliable material with high hydrophobic barrier integrity and resolution. Polypropylene achieved functional microchannels with a resolution of 621 {+/-} 33{micro}m, hydrophobic barrier integrity of (93.75 {+/-} 9.16%), wicking speed of 0.38mm/s and optimal hydrophilicity of channels (51.4 {+/-} 8.36 {degrees}) with minimal embedding during thermal curing. To demonstrate proof of principle, a fluorescence assay demonstrating the formation of a dimeric g-quadruplex structure from a g-rich sequence which significantly enhances fluorescence of thioflavin T was implemented.

It has been recognized a long time ago that the hedgehog (Hh) and Wnt signaling pathways have numerous similarities that suggest their common evolutionary origin. Although the Hh and Wnt proteins are unrelated they are similar in as much as they carry lipid modifications that are critical for their interaction with their receptors. In our earlier work we have shown that Wnt inhibitory factor 1 (WIF1), originally identified as a Wnt antagonist also binds to and inhibits the signaling activity of sonic hedgehog (Shh), raising the possibility that the lipid moieties of these unrelated morphogens play a dominant role in their interaction with WIF1. In the present work we have compared the interactions of human WIF1 protein with lipidated and non-lipidated forms of human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein has significantly higher affinity for lipidated than non-lipidated Shh, indicating that lipid modifications of Hhs are important for interactions with WIF1.

Infectious diseases remain a persistent global health burden, with bacterial infections predominating. The growing global burden of drug-resistant infections has led to greater emphasis on the discovery and development of novel antibacterial compounds. In an attempt to discover new potent antibacterials, the antibacterial activity of novel 2-substituted benzimidazole derivatives (NR-1 to NR-9) was evaluated in this study against three bacteria, viz. M. smegmatis, B. subtilis and E. coli in vitro using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Among the nine derivatives assessed, two (NR-4 and NR-5) exhibited inhibitory activity against M. smegmatis, while two (NR-5 and NR-7) were active against B. subtilis, with MICs between 62.5 and 250 g/ml. Notably, NR-5 demonstrated antibacterial activity against both M. smegmatis and B. subtilis, with more efficacy against M. smegmatis (MIC: 62.5 g/ml), which was considerably closer to rifampicin (MIC: 31.25 g/ml). Cytotoxicity analysis of these derivatives in Vero cells indicated minimal toxicity for NR-4 and NR-5, and SwissADME evaluation suggested favourable physicochemical properties and drug-likeness, supporting good oral bioavailability. Moreover, the growth kinetics profiling of the NR-5 Benzimidazole derivative demonstrated that it inhibited the growth of M. smegmatis effectively, even after prolonged exposure. These findings highlighted the promise of the active benzimidazole derivative, NR-5, as a potential candidate for developing a more effective and less toxic antimycobacterial drug. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/710429v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@126a0fcorg.highwire.dtl.DTLVardef@1131372org.highwire.dtl.DTLVardef@161a70corg.highwire.dtl.DTLVardef@1e0dbe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

Galectin-3 (Gal3) is one of the most pro-inflammatory proteins and a biomarker of inflammatory diseases and cancer. Previous studies showed that Gal3 binds to v and {beta}1 integrins but it is unclear how Gal3 binds to integrins. Here, we show that Gal3 bound to soluble v{beta}3 and IIb{beta}3 integrins in 1 mM Mn2+ in cell-free conditions in a glycan-independent manner. Docking simulation predicts that Gal3 binds to the classical RGD-binding site (site 1) of v{beta}3, but the predicted Gal3-binding site does not include galactose-binding site. RGDfV or eptifibatide inhibited Gal3 binding to v{beta}3 and IIb{beta}3, respectively, but lactose, pan-galectin inhibitor, did not inhibit Gal3 binding to integrins. Point mutations of the predicted site 1 binding interface of Gal3 effectively inhibited Gal3 binding to site 1. Site 2 is involved in pro-inflammatory signaling (e.g., TNF and IL-6 secretion) and we previously showed that pro-inflammatory cytokines (e.g., CCL5 and TNF) bind to site 2 and allosteric integrin activation. Docking simulation predicts that Gal3 binds to site 2 of v{beta}3 and 5{beta}1. We found that Gal3 induced allosteric activation of soluble integrins v{beta}3, IIb{beta}3, and 5{beta}1 in 1 mM Ca2+ in cell-free conditions. Point mutations in the predicted site 2-binding interface inhibited Gal3-induced integrin activation, suggesting that Gal3 binding to site 2 is required for Gal3-induced integrin activation. Known anti-inflammatory agents, Ivermectin, NRG1, and FGF1 inhibited integrin activation induced by Gal3 in v{beta}3 and IIb{beta}3. These findings suggest that Gal3 binding to site 2 may be a potential mechanism of pro-inflammatory and pro-thrombotic action of Gal3.

The interaction between neuronal nitric oxide synthase (NOS1) and its adaptor protein CAPON (NOS1AP) plays a critical role in various neurological processes and has been implicated in cardiovascular and neuropsychiatric disorders. Disruption of this protein-protein interaction represents a potential therapeutic strategy, yet identifying small molecule inhibitors has been challenging. Here, we present the development and validation of a NanoBiT-based luminescence complementation assay optimized for high-throughput screening (HTS) of NOS1-NOS1AP interaction inhibitors. We engineered NOS1 and NOS1AP fusion proteins with HiBiT and LgBiT complementary subunits, respectively, and established stable CHO-K1 cell lines for robust signal generation. The assay demonstrated excellent performance characteristics with a signal-to-background ratio exceeding 240-fold, and was validated using TAT-GESV, a known peptide inhibitor that showed time- and dose-dependent inhibition. We successfully screened a diverse library of 10,240 compounds and identified 19 validated hits with IC50 values ranging from 2.54 to greater than 30 M, with the majority exhibiting IC50 values below 30 M. The top three compounds exhibited potent inhibitory activity with IC50 values of less than 5 M. This NanoBiT-based assay provides a reliable and efficient platform for discovering novel NOS1-NOS1AP interaction inhibitors and can be adapted for other protein-protein interaction studies.

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role. HighlightsO_LIstructure of RNase 2 in complex with mono and disaccharides at atomic resolution C_LIO_LIidentification of RNase 2 unique sugar binding sites C_LIO_LIcharacterization of a mammalian RNase A family enzyme with lectin properties C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713198v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d805f7org.highwire.dtl.DTLVardef@16fcc49org.highwire.dtl.DTLVardef@ccfd92org.highwire.dtl.DTLVardef@1b8f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Fungal pathogens are an escalating global public health concern, particularly in the context of invasive and opportunistic infections. Cryptococcosis, primarily caused by Cryptococcus neoformans var. grubii, can manifest as acute, subacute, or chronic disease, affecting multiple organs and frequently leading to life-threatening meningitis in immunocompromised individuals. Given the limited antifungal therapeutic strategies and the emergence of resistance and toxicity-related constraints, the development of novel anti-cryptococcal agents remains an urgent priority. In this study, a library of innovative hybrids (5a-f) based on the 3-hydroxypyridin-4(1H)-one scaffold was developed. Their antimicrobial activity was evaluated towards a panel of clinically relevant Gram-positive (methicillin-resistant Staphylococcus aureus - MRSA) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii), as well as fungal species Candida albicans and Cryptococcus neoformans var. grubbi. Cytotoxicity was assessed in HEK293 and HepG2 cell lines, and haemolytic profile was determined to evaluate safety. In addition, iron-chelating capacity and lipophilic properties were also investigated. All compounds formed stable complexes with iron(III) and were non-toxic at concentrations up to 25 M. Lipophilicity studies showed that compounds in series 1 (5a-c) exhibited lower lipophilicity than those in Series 2 (5d-f), mainly due to the regioisomeric position of the hydroxyl group on the 2-methyl-4-pyridone scaffold; specifically, the C3-substitution pattern in Series 2 that enhances the hydrophobic character compared to the C5-substitution in Series 1. Fluorination further increased lipophilicity in both series. Notably, compounds 5c-5f emerged as potent, selective, and non-toxic antifungal agents against Cryptococcus neoformans var. grubii (MIC < 16 {micro}g/mL; CC50 > 32 {micro}g/mL; HC10 > 32 {micro}g/mL). Their distinct structural features appear to play a key role in antifungal selectivity, supporting the potential of these 3-hydroxypyridin-4(1H)-one-based hybrids as promising approach for the development of novel therapeutics for cryptococcal meningitis.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

The hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein forms a complex with SMCR8 and WDR41 (CSW), which acts as a GTPase-activating protein (GAP) regulating small GTPases like ARF1 and RABs involved in intracellular trafficking. Although these findings implicated the ARF1 dysregulation in ALS/FTD and the critical need for validation of its inhibition as potential intervention, small molecules that target the interactions between CSW and ARF1 are lacking. In this study, we showed that the tyrosine-phosphorylated form (Tyr-782) of ASAP1, an ARF-GAP that inactivates ARF1, is increased in the motor cortex of both sporadic ALS and ALS with C9orf72 mutations. Overexpression of C9orf72 led to Golgi disorganization, partially mimicking the effects of ARF1 inhibitor brefeldin A on dispersion of Golgi apparatus. To identify a better strategy to enhance C9orf72 and ARF1 interactions, we applied rational design and virtual screening of a 40-million compound library of small molecules targeting the ARF1-CSW interface. Molecular docking, MM-GBSA binding energy, ADME/Tox profiles, and interaction analysis established MCULE-5095997944 as a top candidate for ARF1 modulation. MCULE-5095997944 demonstrated strong binding to ARF1 in the nanomolar range, reduced GTP-bound ARF1 levels upon ARF1 activation, and altered ARF1-dependent Golgi organization. These studies identified the first small molecule targeting ARF1-CSW interaction and further support ARF1 modulation as a potential therapeutic approach for ALS/FTD.

We used an autonomous lab, comprising a large language model (LLM) and a fully automated cloud laboratory, to optimize the cost efficiency of cell-free protein synthesis (CFPS). By conducting iterative optimization, the LLM-driven autonomous lab was able to achieve a 40% reduction in the specific cost ($/g protein) of CFPS relative to the state of the art (SOTA). This cost reduction was accompanied by a 27% increase in protein production titer (g/L). Iterative experimental design, experiment execution, data capture and analysis, data interpretation, and new hypothesis generation were all handled by the LLM-driven autonomous lab. The interface between OpenAIs GPT-5 LLM and Ginkgo Bioworks cloud laboratory incorporated built-in validation checks via a Pydantic schema to ensure that AI-designed experiments were properly specified. Experimental designs were translated into programmatic specification of multi-instrument biological workflows by Ginkgos Catalyst software and executed on Ginkgos Reconfigurable Automation Cart (RAC) laboratory automation platform, with human intervention largely limited to reagent and consumables preparation, loading and unloading. By integrating LLMs with programmatic control of a cloud lab, we demonstrate that an LLM-driven autonomous lab can successfully perform a real-world scientific task, highlighting the potential of AI-driven autonomous labs for scientific advancement.

Splice site mutations represent a major class of pathogenic mutations in many diseases, as these changes disrupt normal splicing leading to gene expression changes. Cystic fibrosis (CF) results from mutations to the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an essential ion channel. Approximately 13% of the over 2,100 known CFTR mutations disrupt 3 or 5 splice sites and are predicted to cause splicing defects. Because each splicing mutation is rare, developing individualized therapies to treat each one is financially challenging. Exon specific U1 snRNA (ExSpeU1) targets the non-conserved intronic region downstream the 5 splice site (ss) to rescue exon skipping. Because this approach is exon-rather than mutation-specific, a single agent can potentially rescue multiple mutations. In this study, we have developed a platform to systematically classify all patient variants associated with an exon that are predicted to affect splicing and then determine their rescue potential using ExSpeU1. Here we report the results of these studies. Our minigene reporter study shows that 7 of 10 exon 18 variants resulted in exon skipping. Four mutations at the 3 and 5 ss were rescued at least partially using a single ExSpeU1. Using a luciferase reporter, we observe that the splicing rescue is reflected at the protein level. Lastly, we demonstrate exon-targeting ExSpeU1s can also rescue 3 and 5 ss mutations. Overall, this study exemplifies the power of our platform to screen and rescue multiple patient-derived splicing mutations using a single agent.

Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (Mpro) is essential for viral replication and is a conserved target among coronaviruses. Most known Mpro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 Mpro. From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 Mpro. Among them, compound 25, a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC50) of 99 M. Additionally, two thiosemicarbazone analogs (compounds 50 and 51) inhibited SARS-CoV-2 Mpro with IC50 values of 61 M and 70 M. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 Mpro. Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 Mpro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 Mpro inhibitors.

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

Prasugrel is a prodrug, widely used in antiplatelet strategy for secondary prevention after acute coronary syndrome. The metabolism of prasugrel leads to the formation of the Prasugrel Active Metabolite (PAM), an irreversible P2Y12 receptor antagonist. Its mode of binding has not yet been fully established, although it is known that it binds covalently to P2Y12 by forming a disulfide bridge with cysteines and its sulfur moiety. PAM is a molecule with two chiral centers, resulting in four stereoisomers which appear to be stereoselective upon binding. A combination of different molecular modeling methods, such as molecular dynamics, ensemble docking, and Density Functional Theory (DFT), were used to rationalize these differences in antagonism observed in vitro and to elucidate the mode of binding of PAM to P2Y12. PAM is found to bind to the closed P2Y12 conformation in a preferential way. Although the four stereoisomers have comparable affinity, the location of the RS stereoisomer makes the formation of a disulfide bond with cysteines more favorable, particularly with cysteine 175. Compared to the RR stereoisomer, the RS stereoisomer interacts less deeply with the P2Y12 receptor, interacting in particular with the second and third extracellular loops, explaining the competition observed with cangrelor and an intermediate metabolite of prasugrel. Furthermore, DFT calculations have shown that the formation of a disulfide bridge is energetically more favorable with the RS stereoisomer than with the RR stereoisomer. The physical interactions and chemical reaction between the RS stereoisomer and the P2Y12 receptor are key factors in explaining the stereoselective binding of PAM to P2Y12.