Identification of potential pharmacological chaperones that selectively stabilize mutated Aspartoacylases in Canavan disease.

Canavan disease is caused by mutations in the ASPA gene, leading to diminished catalytic activity of aspartoacylase in the brain. Clinical missense mutations are found throughout the enzyme structure, with many of these mutated enzymes having not only decreased activity but also compromised stability. High-throughput screening of a small molecule library has identified several compounds that significantly increase the thermal stability of the E285A mutant enzyme, the most predominant clinical mutation in Canavan disease, while having a negligible effect on the native enzyme. Based on the initial successes, some structural analogs of these initial hits were selected for further examination. Glutathione, NAAG and patulin were each confirmed to be competitive inhibitors, indicating the binding of these compounds at the dimer interface or near the active site of the E285A enzyme. The experimental results were theoretically examined with the help of the docking analysis method. The structure activity-guided optimization of these compounds can potentially lead to potential pharmacological chaperones that could alleviate the detrimental effect of ASPA mutations in Canavan patients.

High-throughput detection of potential bacteriocin producers in a large strain library using live fluorescent biosensors.

The global increase in antibiotic resistances demands for additional efforts to identify novel antimicrobials such as bacteriocins. These antimicrobial peptides of bacterial origin are already used widely in food preservation and promising alternatives for antibiotics in animal feed and some clinical setting. Identification of novel antimicrobials is facilitated by appropriate high throughput screening (HTS) methods. Previously, we have described a rapid, simple and cost-efficient assay based on live biosensor bacteria for detection of antimicrobial compounds that act on membrane integrity using the ratiometric pH-dependent fluorescent protein pHluorin2 (pHin2). Here, we use these biosensors to develop an integrated pipeline for high-throughput identification of bacteriocin producers and their biosynthetic gene clusters. We extend the existing portfolio of biosensors by generating pHin2 expressing strains of Escherichia coli, Bacillus cereus, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus. These strains were characterized, and control experiments were performed to assess heterogeneity of these biosensors in response to known bacteriocins and develop a robust HTS system. To allow detection of compounds that inhibit target bacteria by inhibiting growth without disturbing membrane integrity, the HTS system was extended with a growth-dependent readout. Using this HTS system, we screened supernatants of a total of 395 strains of a collection of lactic acid bacteria. After two rounds of screening 19 strains of the collection were identified that produced antimicrobial activity against Listeria innocua and Listeria monocytogenes. Genomes of confirmed hits were sequenced and annotated. In silico analysis revealed that the identified strains encode between one and six biosynthetic gene clusters (BGCs) for bacteriocins. Our results suggest that pHin2 biosensors provides a flexible, cheap, fast, robust and easy to handle HTS system for identification of potential bacteriocins and their BGCs in large strain collections.

Genetically Encoded Microtubule Binders for Single-Cell Interrogation of Cytoskeleton Dynamics and Protein Activity.

Microtubule (MT) dynamics is tightly regulated by microtubule-associated proteins (MAPs) and various post-translational modifications (PTMs) of tubulin. Here, we introduce OligoMT and OligoTIP as genetically encoded oligomeric MT binders designed for real-time visualization and manipulation of MT behaviors within living cells. OligoMT acts as a reliable marker to label the MT cytoskeleton, while OligoTIP allows for live monitoring of the growing MT plus-ends. These engineered MT binders have been successfully utilized to label the MT network, monitor cell division, track MT plus-ends, and assess the effect of tubulin acetylation on the MT stability at the single-cell level. Moreover, OligoMT and OligoTIP can be repurposed as biosensors for quantitative assessment of drug actions and for reporting enzymatic activity. Overall, these engineered MT binders hold promise for advancing the mechanistic dissection of MT biology and have translational applications in cell-based high-throughput drug discovery efforts.

Identification of small molecule antagonists of sonic hedgehog/heparin binding with activity in hedgehog functional assays.

Sonic hedgehog (Shh) is a morphogen with important roles in embryonic development and in the development of a number of cancers. Its activity is modulated by interactions with binding partners and co-receptors including heparin and heparin sulfate proteoglycans (HSPG). To identify antagonists of Shh/heparin binding, a diverse collection of 34,560 chemicals was screened in single point 384-well format. We identified and confirmed twenty six novel small molecule antagonists with diverse structures including four scaffolds that gave rise to multiple hits. Nineteen of the confirmed hits blocked binding of the N-terminal fragment of Shh (ShhN) to heparin with IC50 values <50 µM. In the Shh-responsive C3H10T1/2 cell model, four of the compounds demonstrated the ability to block ShhN-induced alkaline phosphatase activity. To demonstrate a direct and selective effect on ShhN ligand mediated activity, two of the compounds were able to block induction of Gli1 mRNA, a primary downstream marker for Shh signaling activity, in Shh-mediated but not Smoothened agonist (SAG)-mediated C3H10T1/2 cells. Direct binding of the two compounds to ShhN was confirmed by thermal shift assay and molecular docking simulations, with both compounds docking with the N-terminal heparin binding domain of Shh. Overall, our findings indicate that small molecule compounds that block ShhN binding to heparin and act to inhibit Shh mediated activity in vitro can be identified. We propose that the interaction between Shh and HSPGs provides a novel target for identifying small molecules that bind Shh, potentially leading to novel tool compounds to probe Shh ligand function.

Unleashing the potential of cell painting assays for compound activities and hazards prediction.

The cell painting (CP) assay has emerged as a potent imaging-based high-throughput phenotypic profiling (HTPP) tool that provides comprehensive input data for in silico prediction of compound activities and potential hazards in drug discovery and toxicology. CP enables the rapid, multiplexed investigation of various molecular mechanisms for thousands of compounds at the single-cell level. The resulting large volumes of image data provide great opportunities but also pose challenges to image and data analysis routines as well as property prediction models. This review addresses the integration of CP-based phenotypic data together with or in substitute of structural information from compounds into machine (ML) and deep learning (DL) models to predict compound activities for various human-relevant disease endpoints and to identify the underlying modes-of-action (MoA) while avoiding unnecessary animal testing. The successful application of CP in combination with powerful ML/DL models promises further advances in understanding compound responses of cells guiding therapeutic development and risk assessment. Therefore, this review highlights the importance of unlocking the potential of CP assays when combined with molecular fingerprints for compound evaluation and discusses the current challenges that are associated with this approach.

Creating large-scale genetic diversity in Arabidopsis via base editing-mediated deep artificial evolution.

BACKGROUND: Base editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants. RESULTS: In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding. CONCLUSIONS: Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.

Screening for Genes Involved in Outer Membrane Vesicle Biogenesis in Bacteroides thetaiotaomicron.

Bacteroides spp. are prominent gut commensals that are believed to modulate the intestinal environment, in part, by producing outer membrane vesicles (OMVs). Bacteroides OMVs have been ascribed many functions in vitro, but the genetic underpinnings behind OMV biogenesis and regulation are unclear. Understanding the mechanism of OMV biogenesis is required to determine the importance of Bacteroides OMVs in vivo. Here, we describe our methodology for screening Bacteroides thetaiotaomicron VPI-5482 to identify genes required for OMV biogenesis and regulation in a high-throughput format. This protocol is easily adaptable and can potentially be employed to further our knowledge of OMV biogenesis in other bacteria.

A batch screening technique for the calculation of chromatographic separability.

This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.

A Minimalist Method for Fully Oxygen-Tolerant RAFT Polymerization through Sulfur-Centered Trithiocarbonate Radical Initiation.

In recent years, the fully oxygen-tolerant reversible deactivation radical polymerization (RDRP) has become a highly researched area. In this contribution, a new and minimalist method is successfully employed to accomplish fully oxygen-tolerant reversible addition-fragmentation chain transfer (RAFT) polymerization using bis(trithiocarbonate) disulfides (BisTTC) as an iniferter agent, where the released sulfur-centered trithiocarbonate (TTC) radical can initiate monomer. Furthermore, polymerization kinetics revealed the typical "living" features of this polymerization system. More importantly, by high-throughput screening, it is found that dodecyl-substituted TTC is responsible for the fully oxygen-tolerant RAFT polymerization though trithiocarbonate radical initiation and R radical deoxygenation. It is believed that trithiocarbonate radical initiation strategy provides a powerful and minimalist tool for fully oxygen-tolerant RDRPs.

Genetic circuits for metabolic flux optimization.

In recent years, genetic circuit-based regulation of metabolic flux in microbial cell factories has received significant attention. In this review, we describe a pipeline for the design and construction of genetic circuits for metabolic flux optimization. In particular, we summarize the recent advances in computationally assisted prediction of critical metabolic nodes and genetic circuit design automation. Further, we introduce strategies for constructing high-performance genetic circuits. We also summarize the latest applications of genetic circuits in the dynamic regulation of metabolism and high-throughput screening. Finally, we discuss the challenges and prospects associated with the design and construction of sophisticated genetic circuits. Through this review, we aim to provide a theoretical basis for designing and constructing high-performance genetic circuits to optimize metabolic flux.

High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.

Functionalized Polysaccharides Improve Sensitivity of Tyramide/Peroxidase Proximity Labeling Assays through Electrostatic Interactions.

High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.

Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

In-cell bioluminescence resonance energy transfer (BRET)-based assay uncovers ceritinib and CA-074 as SARS-CoV-2 papain-like protease inhibitors.

Papain-like protease (PLpro) is an attractive anti-coronavirus target. The development of PLpro inhibitors, however, is hampered by the limitations of the existing PLpro assay and the scarcity of validated active compounds. We developed a novel in-cell PLpro assay based on BRET and used it to evaluate and discover SARS-CoV-2 PLpro inhibitors. The developed assay demonstrated remarkable sensitivity for detecting the reduction of intracellular PLpro activity while presenting high reliability and performance for inhibitor evaluation and high-throughput screening. Using this assay, three protease inhibitors were identified as novel PLpro inhibitors that are structurally disparate from those previously known. Subsequent enzymatic assays and ligand-protein interaction analysis based on molecular docking revealed that ceritinib directly inhibited PLpro, showing high geometric complementarity with the substrate-binding pocket in PLpro, whereas CA-074 methyl ester underwent intracellular hydrolysis, exposing a free carboxyhydroxyl group essential for hydrogen bonding with G266 in the BL2 groove, resulting in PLpro inhibition.

Discovery of a Peptoid-Based Nanoparticle Platform for Therapeutic mRNA Delivery via Diverse Library Clustering and Structural Parametrization.

Nanoparticle-mediated mRNA delivery has emerged as a promising therapeutic modality, but its growth is still limited by the discovery and optimization of effective and well-tolerated delivery strategies. Lipid nanoparticles containing charged or ionizable lipids are an emerging standard for in vivo mRNA delivery, so creating facile, tunable strategies to synthesize these key lipid-like molecules is essential to advance the field. Here, we generate a library of N-substituted glycine oligomers, peptoids, and undertake a multistage down-selection process to identify lead candidate peptoids as the ionizable component in our Nutshell nanoparticle platform. First, we identify a promising peptoid structural motif by clustering a library of >200 molecules based on predicted physical properties and evaluate members of each cluster for reporter gene expression in vivo. Then, the lead peptoid motif is optimized using design of experiments methodology to explore variations on the charged and lipophilic portions of the peptoid, facilitating the discovery of trends between structural elements and nanoparticle properties. We further demonstrate that peptoid-based Nutshells leads to expression of therapeutically relevant levels of an anti-respiratory syncytial virus antibody in mice with minimal tolerability concerns or induced immune responses compared to benchmark ionizable lipid, DLin-MC3-DMA. Through this work, we present peptoid-based nanoparticles as a tunable delivery platform that can be optimized toward a range of therapeutic programs.

Protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches.

Developing antibodies with high specificity against post-translationally modified epitopes remains a challenge. Yeast biopanning is well suited in screening for high-specificity binders. Here, we present a protocol for screening and validating antibodies specific to protein phosphorylation sites using a set of yeast biopanning approaches. We describe steps for screening a yeast surface display library for antibodies and other binders. We then detail procedures for validating the antibodies found by analyzing their specificity through whole-well image analysis in 96-well plates. For complete details on the use and execution of this protocol, please refer to Arbaciauskaite et al.1.

Large-Scale Computational Screening-Aided Development of High-Performance Adsorbent for Simultaneous Capture of Aromatic Volatile Organic Compounds.

The development of an efficient adsorbent for the simultaneous capture of large amounts of benzene, toluene, ethylbenzene, and xylene isomers (BTEX) is an important and challenging issue. Here, through a stepwise screening of 10,142 metal-organic framework (MOF) structures from the computation-ready, experimental (CoRE) MOF database, 65 MOFs are proposed as promising adsorbent candidates for BTEX capture by considering the structures with accessible pore sizes for BTEX adsorption, sufficient hydrophobicity, high benzene selectivity (>0.2), and large total BTEX uptake (>3 mmol/g). Among the top-performing MOFs in terms of the BTEXmatrix (total BTEX uptake × benzene selectivity), EGUELUY01 was synthesized, and it exhibited large uptakes (≈5 mmol/g) for all BTEX components at concentrations of 1200-1500 ppm, which are superior to the BTEX uptake of the benchmark adsorbent, activated carbon. Moreover, some structure-property relationships required for BTEX adsorbents are provided through the obtained large-scale simulation data and machine learning analysis. The determined relationships will be useful for the future development of efficient BTEX adsorbents.

Identification of Ribonuclease Inhibitors for the Control of Pathogenic Bacteria.

Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials.

Typical emerging contaminants in sewage treatment plant effluent, and related watersheds in the Pearl River Basin: Ecological risks and source identification.

Emerging contaminants pose a potential risk to aquatic ecosystems in the Pearl River Basin, China, owing to the high population density and active industry. This study investigated samples from eight sewage treatment plants, and five surface water bodies of related watersheds. To screen the risk of emerging contaminants (ECs), and clarify their sources, this study calculated the risk quotient of detected chemical and performed source identification/apportionment using the positive matrix factorization method. In total, 149 organic pollutants were identified. Pharmaceuticals showed significant concentrations in sewage treatment plant samples (120.87 ng/L), compared with surface water samples (1.13 ng/L). The ecological risk assessment identified three chemicals with a heightened risk to aquatic organisms: fipronil sulfide, caffeine, and roxithromycin. Four principal sources of contaminants were identified: pharmaceutical wastewater, domestic sewage, medical effluent, and agricultural runoff. Pharmaceutical wastewater was the primary contributor (60.4 %), to the cumulative EC concentration and to ECs in sewage treatment plant effluent. Agricultural drainage was the main source of ECs in surface water. This study provides a strategy to obtain comprehensive information on the aquatic risks and potential sources of EC species in areas affected by artificial activities, which is of substantial importance to pollutant management and control.

Identification of novel neuroprotectants against vincristine-induced neurotoxicity in iPSC-derived neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of cancer chemotherapy that can often limit treatment options for cancer patients or have life-long neurodegenerative consequences that reduce the patient's quality of life. CIPN is caused by the detrimental actions of various chemotherapeutic agents on peripheral axons. Currently, there are no approved preventative measures or treatment options for CIPN, highlighting the need for the discovery of novel therapeutics and improving our understanding of disease mechanisms. In this study, we utilized human-induced pluripotent stem cell (hiPSC)-derived motor neurons as a platform to mimic axonal damage after treatment with vincristine, a chemotherapeutic used for the treatment of breast cancers, osteosarcomas, and leukemia. We screened a total of 1902 small molecules for neuroprotective properties in rescuing vincristine-induced axon growth deficits. From our primary screen, we identified 38 hit compounds that were subjected to secondary dose response screens. Six compounds showed favorable pharmacological profiles - AZD7762, A-674563, Blebbistatin, Glesatinib, KW-2449, and Pelitinib, all novel neuroprotectants against vincristine toxicity to neurons. In addition, four of these six compounds also showed efficacy against vincristine-induced growth arrest in human iPSC-derived sensory neurons. In this study, we utilized high-throughput screening of a large library of compounds in a therapeutically relevant assay. We identified several novel compounds that are efficacious in protecting different neuronal subtypes from the toxicity induced by a common chemotherapeutic agent, vincristine which could have therapeutic potential in the clinic.