ACS Omega

Scar formation is a process that occurs due to increased collagen deposition and uncontrolled inflammation. Previous studies have demonstrated that Pirfenidone (Pf), an FDA approved anti-inflammatory and anti-fibrotic drug can reduce inflammation in vivo as well as regulate activation of LPS-stimulated neutrophils. However, the molecular level mechanism of Pfs action is not well understood. Here, we used neural networks to identify new targets and molecular modeling methods to investigate the Pfs action pathways at the molecular level that are related to its ability to reduce both the inflammatory and remodeling phases of the wound healing process. Out of all the potential targets identified, both molecular docking and molecular dynamics results suggest that Pf has a noteworthy binding preference towards the active conformation of the p38 mitogen activated protein kinase-14 (MAPK14) and it is potentially a type I inhibitor-like molecule. In addition to p38 MAPK (MAPK14), additional potential targets of Pf include AKT1, MAP3K4, MAP2K3, MAP2K6, MSK2, MAP2K2, ERK1, ERK2, and PDK1. We conclude that several proteins/kinases, rather than a single target, are involved in Pfs wound healing ability to regulate signaling, inflammation, and proliferation.

Nanowires (NW) have been extensively studied for Shewanella spp. and Geobacter spp. and are mostly produced by Type IV pili or multiheme c-type cytochrome. Electron transfer via NW is the most studied mechanism in microbially induced corrosion, with recent interest in application in bioelectronics and biosensor. In this study, a machine learning (ML) based tool was developed to classify NW proteins. A manually curated 999 protein collection was developed as an NW protein dataset. Gene ontology analysis of the dataset revealed microbial NW is part of membranal proteins with metal ion binding motifs and plays a central role in electron transfer activity. Random Forest (RF), support vector machine (SVM), and extreme gradient boost (XGBoost) models were implemented in the prediction model and were observed to identify target proteins based on functional, structural, and physicochemical properties with 89.33%, 95.6%, and 99.99% accuracy. Dipetide amino acid composition, transition, and distribution protein features of NW are key important features aiding in the models high performance.

Chemo-resistance remains the main hurdle to cancer therapy, challenging the improvement of clinical outcomes in cancer patients. Therefore, exploratory studies to address chemo-resistance through various approaches are highly rewarding. Nanomedicine is a promising recent advancement in this direction. Comprehensive studies to understand the precise molecular interactions of nanomaterials is necessary to validate their specific "nano induced" effects. Here, we illustrate in detail the specific biological interactions of vanadium pentoxide nanoparticles (VnNp) on triple-negative breast cancer cells and provide initial insights towards its potential in breast cancer management at the cellular level. VnNp shows a time-dependent anti-oxidant and pro-oxidant property in vitro. These nanoparticles specifically accumulate in the lysosomes and mitochondria, modulate various cellular processes including impaired lysosomal function, mitochondrial damage, and induce autophagy. At more extended periods, VnNp influences cell cycle arrest and inhibits cell migration potentiating the onset of apoptosis. Preliminary in vivo studies, on exposing healthy Swiss albino mice to VnNp demonstrated normal blood parameters, organ distribution, and tissue redox balance which further indicated the absence of any adverse organ toxicity. Hence, we foresee tumor-targeting VnNp as a potential drug molecule for future cancer management.

There is great need for novel strategies to tackle antimicrobial resistance, in particular in Gram-negative species such as Escherichia coli that cause opportunistic infections of already compromised patients. Here we demonstrate, following a screen of G-quadruplex (G4) ligand candidates, that a novel pyridinium-functionalized azobenzene L9 shows promising antibacterial activity (MIC values [≤] 4 g/mL) against multi-drug resistant E. coli. Tandem Mass Tag (TMT) proteomics of E. coli treated with sub-lethal concentrations of L9, identified that, consistent with its superior antibacterial activity, L9 treatment influences expression levels of more G4-associated proteins than the analogous ligands L5 (stiff-stilbene) or pyridostatin (PDS), and upregulates multiple essential proteins involved in translation. Biophysical analysis showed L9 binds potential target G4-containing sequences, identified from proteomic experiments and by bioinformatics, with variable affinity, in contrast to the two comparator G4 ligands (L5, PDS) that better stabilize G4 structures but have lower antimicrobial activity. Fluorescence microscopy-based Bacterial Cytological Profiling (BCP) suggests that the L9 mechanism of action is distinct from other antibiotic classes. These findings support strategies discovering potential G4 ligands as antibacterial candidates for priority targets such as multi-drug resistant E. coli, warranting their further exploration as potential novel therapeutic leads with G4-mediated modes of action. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/506212v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@18197a0org.highwire.dtl.DTLVardef@109b120org.highwire.dtl.DTLVardef@14be8eeorg.highwire.dtl.DTLVardef@a97048_HPS_FORMAT_FIGEXP M_FIG C_FIG

Affinity based drug delivery mechanisms increase efficacy and minimalize off target effects when compared to non-specific methods due to the localization of drugs within target areas. While this is beneficial for targeted delivery, introduction of foreign polymeric medical devices into the body provide a potential area of localization due to high affinity between administered drugs and polymers. Previous attempts at creating models to predict affinity between small molecule drugs and polymers require a specific model be trained for each individual polymer failing to incorporate input features of both the polymer (host) and small molecule drug (guest). Within, we propose a universal model built using a neural network and quantitative structure activity relationships to predict the binding energy between guest and host molecules using input features. The trained model returned a correlation value, R2, of 0.9806 and 0.9958 between predicted and experimental binding affinity for the training and validation sets, respectively. This correlates to a mean absolute error of 0.951 kJ/mol and 0.771 kJ/mol for the training and validation sets, respectively. While limited to the current polymers used to train the model, the dataset can be expanded, and models retrained for further applications.

BackgroundRising antimicrobial resistance rates, require new therapeutic approaches such as antimicrobial peptides (AMPs), which are part of the innate immune defense, as alternatives to antibiotics. In this study, we aim to unravel the antibacterial activity of human histone H1.2 peptide against Pseudomonas aeruginosa and its potential immune modulatory role. MethodsWe used a hemofiltrate peptide database for antimicrobial peptide prediction to identify novel human AMPs. Thirteen sequences of histone H1 were identified as putative AMPs, synthesized, and tested against bacterial ESKAPE pathogens in a radial diffusion assay. SYTOX green assay, electrophoretic mobility shift assay, and differential proteomics assays were conducted to determine the mode of action of H1.2 peptide fragment. A crystal violet assay was performed to evaluate the inhibition of biofilm formation. The cytotoxicity of the peptide was tested in LDH and Alamar assays. Finally, to visualize the contributions of H1.2 in NETs formation, scanning electron microscopy was performed. ResultsThe H1.2 peptide inhibited the growth of P. aeruginosa in a dose and pH-dependent manner without cytotoxicity towards mammalian THP-1 cells. It acts on intracellular targets to inhibit the growth of P. aeruginosa. STRING analysis from the differential proteomics assay showed that H1.2 targets the downregulation of proteins involved in the biogenesis of outer membrane proteins, including the folding and trafficking of outer membrane proteins across the cytoplasmic membrane. Scanning electron microscopy images showed that H1.2 forms NET-like structures capable of trapping and immobilizing P. aeruginosa. ConclusionThe characterized antimicrobial activity of H1.2 points to a role for human histone H1 fragments in innate immunity and may represent a promising approach for the development of novel antibacterial therapies. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/724237v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1778ddborg.highwire.dtl.DTLVardef@26430org.highwire.dtl.DTLVardef@ffbfa2org.highwire.dtl.DTLVardef@7e38ae_HPS_FORMAT_FIGEXP M_FIG C_FIG Sec transport and BAM complex system including chaperone proteins and quality control proteases are inhibited by H1.2 in Pseudomonas aeruginosa.Outer membrane proteins (OMPs) are synthesized in the cytoplasm and transported across the inner membrane via the Sec translocase, assisted by SecA/SecB or ribosomes. In the periplasm, they are escorted by chaperones such as SurA to the BAM complex for insertion into the outer membrane. Here, we show that H1.2, an antimicrobial peptide, targets membrane biogenesis in P. aeruginosa through downregulating Sec translocase (SecA/SecB and SecYEG), SurA, and BAM complex. Therefore, leading to improper transfer, folding and insertion of OMPs into the outer membrane. Normally, misfolded proteins are degraded by the protease MucD to prevent toxic aggregation in the bacteria. However, with H1.2 inhibiting MucD the proteotoxic stress is exacerbated, ultimately compromising bacterial homeostasis and viability. Figure created using BioRender.com.

Platinum-based drugs, such as cisplatin, are first-line chemotherapy treatments for patients with cancer. However, the success of these drugs is balanced with severe off-target toxicities and high dosing requirements, prompting the development of selective nanocarriers for targeted drug delivery. This study uses a computationally guided approach to examine the role of amino acids in cisplatin binding within proteins as nanocarriers. Using density functional theory, we quantify the binding of cisplatin to platinum-coordinating amino acids. We then rationally engineer a model MSH6 protein carrier, and evaluate the ability of MSH6 to bind cisplatin via molecular docking simulations. Structure predictions of the engineered MSH6 show that inserting the cisplatin-binding site has a limited impact on the nearby protein architecture of MSH6. Finally, we confirm and reveal cisplatins mechanism of action with DNA binding, and compare the energetic potentials of DNA binding from protein-delivered cisplatin to systemically administered cisplatin. Future studies will use these results to experimentally validate the binding of cisplatin in model protein carriers, and inform the strategic design and experimental development of a protein nanocarrier to achieve targeted drug delivery in cancer. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/699809v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@feee4org.highwire.dtl.DTLVardef@ccc34org.highwire.dtl.DTLVardef@328cd3org.highwire.dtl.DTLVardef@bb8335_HPS_FORMAT_FIGEXP M_FIG C_FIG

Inhibitors of DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug discovery. They are also chemical tools to understand the biochemistry of epigenetic processes. Herein, we report five distinct inhibitors of DNMT1 characterized in enzymatic inhibition assays that did not show activity with DNMT3B. It was concluded that the dietary component theaflavin is an inhibitor of DNMT1. Two additional novel inhibitors of DNMT1 are the approved drugs glyburide and panobinostat. The DNMT1 enzymatic inhibitory activity of panobinostat, a known pan inhibitor of histone deacetylases, agrees with experimental reports of its ability to reduce DNMT1 activity in liver cancer cell lines. Molecular docking of the active compounds with DNMT1, and re-scoring with the recently developed Extended Connectivity Interaction Features approach, had an excellent agreement between the experimental IC50 values and docking scores.

The possibility of using RNA-targeting small molecules to treat diseases is gaining traction as the next frontier of drug discovery and development. The chemical characteristics of small molecules that bind to RNA are still relatively poorly understood, particularly in comparison to protein-targeting small molecules. To fill this gap, we have generated an unprecedented amount of RNA-small molecule binding data, and used it to derive physicochemical rules of thumb that could be used to define areas of chemical space enriched for RNA binders - the Small molecules Targeting RNA (STaR) rules of thumb. These rules have been applied to publicly available RNA-small molecule datasets and found to be largely generalizable. Furthermore, a number of patented RNA-targeting compounds and FDA-approved compounds also pass these rules, as well as key RNA binding approved drug case studies including Risdiplam. We anticipate this work will significantly accelerate the exploration of the RNA-targeted chemical space, towards unlocking RNAs potential as a small molecule drug target. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=130 HEIGHT=200 SRC="FIGDIR/small/578268v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@15ced07org.highwire.dtl.DTLVardef@1cd891eorg.highwire.dtl.DTLVardef@e51599org.highwire.dtl.DTLVardef@1ecfa86_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.

Amyloid fibril formation is a hallmark of several protein misfolding diseases, including systemic hereditary amyloidosis (SHA), in which lysozyme aggregates into plaques, causing inflammation in various tissues. SHA is a rare disease with no current drug treatment options. In our efforts to identify potential therapeutics for SHA, we investigated the inhibitory effects of benzamidine (BEN) on the fibrillation of human lysozyme (HL). Multiple biophysical assays demonstrated BENs ability to effectively prevent amyloid formation. Intrinsic fluorescence measurements highlighted BENs interaction with HL. We inferred the binding mode of BEN to HL through ITC experiments, molecular docking, and molecular dynamics simulations, confirmed BENs binding at the active site, particularly near stretch-2 (residues 52-64), a key region in its anti-amyloidogenic activity. This interaction differed from the previously reported interaction with HEWL. Further, microscopy analyses, including scanning electron microscopy (SEM) and transmission electron microscopy (TEM), further supported these findings by showing reduced fibril formation and alterations in fibril morphology in the presence of BEN. Importantly, BEN exhibited no cytotoxic effects in HEK-293 cells, reinforcing its potential as a therapeutic candidate for amyloidosis. These results provide strong evidence of BENs anti-amyloidogenic activity and offer a foundation for future drug development targeting lysozyme amyloidosis.

We performed a thorough analysis and characterization of multiple batches of Helaina recombinant human lactoferrin (rhLF, Effera) expressed at an industrial scale in a yeast system. Bottom-up LC-MS/MS-based proteomics analysis detected the full sequence of Helaina rhLF protein and confirmed that its amino acid sequence is identical to that of native human LF (Uniprot i.d. P02788). Helaina rhLF had a protein purity of 98% or higher as determined by three orthogonal methods; reversed-phase HPLC, SDS-PAGE, and LC-MS proteomics analysis. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642. The identified N-glycans of Helaina rhLF were predominantly oligomannose structures with five to nine mannoses (M5-M9), which we also report to be present in both the native human and bovine LF. human milk LF (hmLF) possessed lower levels of oligomannose structures and were mainly M5 and M6. Helaina rhLF protein secondary structure was nearly identical to that of hmLF, as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7{+/-}0.9 {degrees}C and 91.9{+/-}0.5 {degrees}C, consistently mirroring denaturation temperatures observed for apo-and holo-hmLF. Overall, the characterization analysis results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

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.

Alzheimers disease (AD) is characterized by progressive neurodegeneration. The critical molecular trigger is believed to be the accumulation of A{beta} neurotoxic oligomers. Given the proteolytic processing of Amyloid Precursor Protein (APP) by {beta}-secretase (beta-site APP cleaving enzyme 1, BACE1) as the key step in the building up of A{beta} oligomers, BACE inhibitors come with therapeutic prospects of preventing or delaying the onset of Alzheimers. To find inhibitory peptides against BACE1, a library of dark peptides was constructed from 4400 intergenic DNA sequences of Escherichia coli. The sequence level analysis was followed by protein structure predictions, molecular docking, and simulation. Based on bioinformatics analysis, 5 potential peptides were screened for experimental validation. Out of these two peptides were identified as lead molecules based on BACE1 inhibitory activity, followed by FRET inhibitory assay, western blot, and RT-PCR. An 86.7 % drop in BACE1 level was observed in the presence of the ECOI2 peptide. Though encouraging results were obtained from in-silico and in-vitro studies, more work is required to study the efficacy of these peptides in suitable animal models.

Summary/AbstractOdorant binding proteins, OBPs, are a diverse family of small, globular, extra-cellular proteins solubilize volatile organic compounds (VOCs) so they can be internalized and transported by an organism. Since their initial discovery in the early eighties 1, thousands of OBPs have been identified through genome sequencing and characterized by fluorescence ligand binding assays 2. While individual OBPs have been studied in the context of their roles in specific organism, there have been no studies towards the understanding of the comparative structure-function relations of all known OBPs, primarily due to a lack of a centralized database that incorporates the binding affinity with the structure of all OBPs. Incorporating OBP information into a database requires not only an extensive search of all existing resources, but also creating a useful platform that relates sequence structures to target functions. Combining 215 functional studies containing 381 unique OBPs from 91 insect species we created a database, iOBPdb: https://iobpdb.herokuapp.com, of OBP binding affinities for a wide range of VOC targets. We demonstrate here that the construction of this initial database provides powerful search and associative capabilities including interrogating odor binding proteins as clusters and groups by sequence similarity versus protein and target molecular weights, and by the functional groups of the VOC targets. The comparative interrogation of the probe-target recognition allows for a more comprehensive understanding of the underlying structural features of all OBPs that had not been possible by only examining the OBPs individually. We present our results in a variety of phylogenetic representations as well as providing the binding profiles of OBP groups to VOC functional moieties. Potential applications include development of molecular probes for biosensors, novel bioassays and drugs, discovery of novel pesticides which inhibit VOC / OBP interactions, as well providing a foundational basis for the functional understanding of odor sensing and perception in the brain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/498339v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@9c2fa0org.highwire.dtl.DTLVardef@1fad38aorg.highwire.dtl.DTLVardef@56175forg.highwire.dtl.DTLVardef@189797_HPS_FORMAT_FIGEXP M_FIG C_FIG

Synthetic opioids such as fentanyl and related analogs have been widely used for pain management. However, their negative side effects, including respiratory depression and high potential for addiction, underscore the need for a deeper understanding of fentanyls interactions with proteins throughout the human body. Fentanyl analogs bind and activate opioid receptors in the central and peripheral nervous systems, triggering numerous downstream signaling pathways. Increasingly, fentanyl has been shown to interact with non-opioid receptors, and elucidation of these non-canonical fentanyl-protein interactions may provide insights into the mechanisms contributing to fentanyls adverse effects and illuminate novel countermeasure strategies. To identify proteins in mammalian tissues that may interact with fentanyl, we designed and synthesized three affinity-based probes (AfBPs) that include the fentanyl core and feature a diazirine photoaffinity group and alkyne handle for click chemistry at different positions. Molecular docking simulations predicted that these AfBPs bind the mu opioid receptor similarly to fentanyl. Affinity-based protein profiling using the FA-T1 probe in vitro in tissues from six animal species identified histamine N-methyltransferase (HNMT), endophilin-B1 (SH3GLB1), fructosamine-3-kinase (FN3K), cutA divalent cation tolerance analog (CUTA), and monoamine oxidase B (MAOB) among the top proteins that bind fentanyl in multiple species and tissue types. Molecular docking of fentanyl and remifentanil with these protein structures identified putative binding sites. The interaction of fentanyl with specific proteins was empirically assessed through protein structural analyses. These findings highlight potential fentanyl-protein interactions that may contribute to the acute and long-term impacts of fentanyl exposures.

Emerging fungal pathogens represent a concerning threat to both global health and food security. In this study, we aimed to address our rising vulnerability to fungal pathogens through the development of the Fung-AI pipeline: an AI/ML-driven approach for antifungal discovery. A generative adversarial network (GAN) was trained to generate novel candidate antifungal peptide sequences. Next, in silico antifungal and hemolytic classifiers were built to further prioritize AI-generated peptides for experimental validation. From a pool of [~]10,000 candidates, thirteen peptides were selected for testing over two-stages of experimentation. Five peptides were found to display mild antifungal activity against the wheat pathogen, Fusarium graminearum, with minimal inhibitory concentrations (MICs) ranging from 250 {micro}g/mL to 500 {micro}g/mL. Four of the five peptides also showed activity against the human pathogen, Candida albicans (MIC: 500 {micro}g/mL). Two of our AI-generated antifungal peptides additionally demonstrated low cytotoxicity in HepG2 human liver carcinoma cells (LC50 > 704.2 {micro}g/mL) indicating that they may be useful as scaffolds for future optimization for therapeutic applications. None of our peptides were found to considerably inhibit the emerging pathogen C. auris, suggesting the need for pathogen-specific down-selection of candidate peptides. Overall, we present a proof-of-principle, generative-AI-based approach for the rapid design of de novo antifungal peptides.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA approved small molecule inhibitors, to identify those that prevent NDU1-associated functions. We identified an anti-helminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug resistant C. albicans and C. auris from their growth surface. To overcome the sub-optimal solubility and permeability of NCL that is well-known to affect its in vivo efficacy, we developed NCL encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, that also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans, and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms, and uses nanoparticles of an FDA approved non-toxic drug to improve biofilm penetrability and microbial killing.

The carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) serves as a critical regulatory protein controlling nitric oxide (NO) signaling across multiple physiological and pathological processes which encompass neurological, cardiac and metabolic functions. These diverse physiological roles of CAPON marks it as a key therapeutic target for conditions associated with its dysregulation. Despite this therapeutic potential there are no specific CAPON or nNOS/CAPON modulators which have been developed to date, highlighting a significant gap in targeted drug discovery. Herein, we report the first strategy specifically focused on disrupting the nNOS/CAPON protein-protein interface. Through screening of chemical libraries composed of 4.6 million compounds and eight molecular dynamics simulations, two potential hit compounds were identified. Beyond identifying these promising hits, our approach introduces two novel computational tools: a freely available Python-based toolset for NMR structural analysis and visualization and a second toolkit for accelerated ligand preparation. These tools significantly accelerate data preparation timelines while reducing computational costs, providing the research community with accessible resources for structure-based drug discovery efforts. Together, these tools represent a substantial contribution to the computational chemistry toolkit, enabling researchers to conduct high-throughput virtual screening campaigns more efficiently and with greater reproducibility. This work represents a foundational step toward developing targeted therapies for CAPON-mediated disorders and provides a scalable computational framework for future protein-protein interaction drug discovery efforts. HighlightsO_LIA novel structure-based strategy developed to target the CAPON/nNOS protein-protein interaction. C_LIO_LIA Python-based toolset for protein conformation analysis, identification, visualization and separation. C_LIO_LIPython pipeline enables efficient ligand preparation for ultra-large chemical libraries. C_LIO_LIVirtual screening identified 6 promising small-molecule candidates. C_LIO_LIA total of 8*100ns molecular dynamics (MD) simulations performed using DESMOND. C_LIO_LIMM/GBSA and contact-time analysis were conducted to assess binding stability and affinity. C_LI

BackgroundCancer remains a critical global health concern. Among its various forms, colorectal cancer (CRC) stands out due to its high prevalence and mortality rates, emphasizing the urgent need for novel therapeutic agents to enhance treatment efficacy and prolong patient survival. Monastrol, an antimitotic compound known to bind kinesin Eg5, is employed in some cancer therapies. Recent studies have revealed that monastrol also interacts with fascin, a protein implicated in tumor aggressiveness and metastasis, thereby disrupting microtubule dynamics and actin bundling, ultimately impairing cell migration. MethodsIn this work, we developed a workflow to identify fascin-binding compounds based on a monastrol-derived pharmacophore model, integrating in silico predictions with in vitro validation. We performed ligand-based virtual screening using a pharmacophore model constructed from monastrol, applied to a high-throughput screening (HTS) library of 1.6 million compounds. The top-ranking candidates from the virtual screening were subsequently subjected to physicochemical characterization and cellular assays. ResultsTwo compounds (designated Z118298144 and Z17544625) were identified that exhibited strong binding to fascin and inhibited actin bundling in physicochemical assays. Furthermore, cellular experiments demonstrated that both compounds reduced proliferation and impaired migration of CRC cells at micromolar concentrations. ConclusionsWe established an optimized pipeline combining virtual screening with experimental validation to efficiently identify fascin inhibitors. Using this approach, we discovered two promising compounds with anticancer activity in CRC cell cultures. Moreover, the protocol has been successfully adapted for application to additional cancer-related targets, expanding its potential utility in drug discovery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/667829v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@dc470eorg.highwire.dtl.DTLVardef@1bd0759org.highwire.dtl.DTLVardef@12d755dorg.highwire.dtl.DTLVardef@1742d45_HPS_FORMAT_FIGEXP M_FIG C_FIG