From the perspective of experimental practice: High-throughput computational screening in photocatalysis

Photocatalysis,a critical strategy for harvesting sunlight to address energy demand and environmental concerns,is underpinned by the discovery of high-performance photocatalysts,thereby how to design photocatalysts is now generating widespread interest in boosting the conversion effi-ciency of solar energy.In the past decade,computational technologies and theoretical simulations have led to a major leap in the development of high-throughput computational screening strategies for novel high-efficiency photocatalysts.In this viewpoint,we started with introducing the challenges of photocatalysis from the view of experimental practice,especially the inefficiency of the traditional“trial and error”method.Sub-sequently,a cross-sectional comparison between experimental and high-throughput computational screening for photocatalysis is presented and discussed in detail.On the basis of the current experimental progress in photocatalysis,we also exemplified the various challenges associated with high-throughput computational screening strategies.Finally,we offered a preferred high-throughput computational screening procedure for pho-tocatalysts from an experimental practice perspective(model construction and screening,standardized experiments,assessment and revision),with the aim of a better correlation of high-throughput simulations and experimental practices,motivating to search for better descriptors.

Interpretable Machine Learning-Assisted High-Throughput Screening for Understanding NRR Electrocatalyst Performance Modulation between Active Center and C-N Coordination

Understanding the correlation between the fundamental descriptors and catalytic performance is meaningful to guide the design of high-performance electrochemical catalysts.However,exploring key factors that affect catalytic performance in the vast catalyst space remains challenging for people.Herein,to accurately identify the factors that affect the performance of N2 reduction,we apply interpretable machine learning(ML)to analyze high-throughput screening results,which is also suited to other surface reactions in catalysis.To expound on the paradigm,33 promising catalysts are screened from 168 carbon-supported candidates,specifically single-atom catalysts(SACs)supported by a BC_(3)monolayer(TM@V_(B/C)-N_(n)=_(0-3)-BC_(3))via high-throughput screening.Subsequently,the hybrid sampling method and XGBoost model are selected to classify eligible and non-eligible catalysts.Through feature interpretation using Shapley Additive Explanations(SHAP)analysis,two crucial features,that is,the number of valence electrons(N_(v))and nitrogen substitution(N_(n)),are screened out.Combining SHAP analysis and electronic structure calculations,the synergistic effect between an active center with low valence electron numbers and reasonable C-N coordination(a medium fraction of nitrogen substitution)can exhibit high catalytic performance.Finally,six superior catalysts with a limiting potential lower than-0.4 V are predicted.Our workflow offers a rational approach to obtaining key information on catalytic performance from high-throughput screening results to design efficient catalysts that can be applied to other materials and reactions.

High-throughput computational screening and in vitro evaluation identifies 5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]-1H-isoindole-1,3(2H)-dione(C3),as a novel EGFR—HER2 dual inhibitor in gastric tumors

Gastric cancers are caused primarily due to the activation and amplification of the EGFR or HER2 kinases resulting in cell proliferation,adhesion,angiogenesis,and metastasis.Conventional therapies are ineffective due to the intra-tumoral heterogeneity and concomitant genetic mutations.Hence,dual inhibition strategies are recommended to increase potency and reduce cytotoxicity.In this study,we have conducted computational high-throughput screening of the ChemBridge library followed by in vitro assays and identified novel selective inhibitors that have a dual impediment of EGFR/HER2 kinase activities.Diversity-based High-throughput Virtual Screening(D-HTVS)was used to screen the whole ChemBridge small molecular library against EGFR and HER2.The atomistic molecular dynamic simulation was conducted to understand the dynamics and stability of the protein-ligand complexes.EGFR/HER2 kinase enzymes,KATOIII,and Snu-5 cells were used for in vitro validations.The atomistic Molecular Dynamics simulations followed by solvent-based Gibbs binding free energy calculation of top molecules,identified compound C3(5-(4-oxo-4H-3,1-benzoxazin-2-yl)-2-[3-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]-1H-isoindole-1,3(2H)-dione)to have a good affinity for both EGFR and HER2.The predicted compound,C3,was promising with better binding energy,good binding pose,and optimum interactions with the EGFR and HER2 residues.C3 inhibited EGFR and HER2 kinases with IC50 values of 37.24 and 45.83 nM,respectively.The GI50 values of C3 to inhibit KATOIII and Snu-5 cells were 84.76 and 48.26 nM,respectively.Based on these findings,we conclude that the identified compound C3 showed a conceivable dual inhibitory activity on EGFR/HER2 kinase,and therefore can be considered as a plausible lead-like molecule for treating gastric cancers with minimal side effects,though testing in higher models with pharmacokinetic approach is required.

High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

An integrated microfluidics platform with high-throughput single-cell cloning array and concentration gradient generator for efficient cancer drug effect screening

Background:Tumor cell heterogeneity mediated drug resistance has been recognized as the stumbling block of cancer treatment.Elucidating the cytotoxicity of anticancer drugs at single-cell level in a high-throughput way is thus of great value for developing precision therapy.However,current techniques suffer from limitations in dynamically characterizing the responses of thousands of single cells or cell clones presented to multiple drug conditions.Methods:We developed a new microfluidics-based“SMART”platform that is Simple to operate,able to generate a Massive single-cell array and Multiplex drug concentrations,capable of keeping cells Alive,Retainable and Trackable in the microchambers.These features are achieved by integrating a Microfluidic chamber Array(4320 units)and a sixConcentration gradient generator(MAC),which enables highly efficient analysis of leukemia drug effects on single cells and cell clones in a high-throughput way.Results:A simple procedure produces 6 on-chip drug gradients to treat more than 3000 single cells or single-cell derived clones and thus allows an efficient and precise analysis of cell heterogeneity.The statistic results reveal that Imatinib(Ima)and Resveratrol(Res)combination treatment on single cells or clones is much more efficient than Ima or Res single drug treatment,indicated by the markedly reduced half maximal inhibitory concentration(IC50).Additionally,single-cell derived clones demonstrate a higher IC_(50) in each drug treatment compared to single cells.Moreover,primary cells isolated from two leukemia patients are also found with apparent heterogeneity upon drug treatment on MAC.Conclusions:This microfluidics-based“SMART”platform allows high-throughput single-cell capture and culture,dynamic drug-gradient treatment and cell response monitoring,which represents a new approach to efficiently investigate anticancer drug effects and should benefit drug discovery for leukemia and other cancers.

High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.

Machine Learning-Assisted High-Throughput Virtual Screening for On-Demand Customization of Advanced Energetic Materials

Finding energetic materials with tailored properties is always a significant challenge due to low research efficiency in trial and error.Herein,a methodology combining domain knowledge,a machine learning algorithm,and experiments is presented for accelerating the discovery of novel energetic materials.A high-throughput virtual screening(HTVS)system integrating on-demand molecular generation and machine learning models covering the prediction of molecular properties and crystal packing mode scoring is established.With the proposed HTVS system,candidate molecules with promising properties and a desirable crystal packing mode are rapidly targeted from the generated molecular space containing 25112 molecules.Furthermore,a study of the crystal structure and properties shows that the good comprehensive performances of the target molecule are in agreement with the predicted results,thus verifying the effectiveness of the proposed methodology.This work demonstrates a new research paradigm for discovering novel energetic materials and can be extended to other organic materials without manifest obstacles.

A Cell-based High-throughput Screening Assay for Farnesoid X Receptor Agonists

Objective To develop a high-throughput screening assay for Farnesoid X receptor （FXR） agonists based on mammalian one-hybrid system （a chimera receptor gene system） for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain （LBD） was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain （DBD） of yeast GAL4 of pBIND to construct a GAL4-FXR （LBD） chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD （189-472） chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z＇ factor value of 0.65, Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

High-Throughput Screening Using Fourier-Transform Infrared Imaging

Efficient parallel screening of combinatorial libraries is one of the most challenging aspects of the high- throughput （HT） heterogeneous catalysis workflow. Today, a number of methods have been used in HT catalyst studies, including various optical, mass-spectrometry, and gas- chromatography techniques. Of these, rapid-scanning Fourier-transform infrared （FTIR） imaging is one of the fastest and most versatile screening techniques. Here, the new design of the 16-channel HT reactor is presented and test results for its accuracy and reproducibility are shown. The performance of the system was evaluated through the oxidation of CO over commercial Pd/AI203 and cobalt oxide nanoparticles synthesized with different reducer-reductant molar ratios, surfactant types, metal and surfactant concentrations, synthesis temperatures, and ramp rates.

High-throughput computational screening and design of nanoporous materials for methane storage and carbon dioxide capture

The globally increasing concentrations of greenhouse gases in atmosphere after combustion of coal-or petroleum-based fuels give rise to tremendous interest in searching for porous materials to efficiently capture carbon dioxide(CO_2) and store methane(CH4), where the latter is a kind of clean energy source with abundant reserves and lower CO_2 emission. Hundreds of thousands of porous materials can be enrolled on the candidate list, but how to quickly identify the really promising ones, or even evolve materials(namely, rational design high-performing candidates) based on the large database of present porous materials? In this context, high-throughput computational techniques, which have emerged in the past few years as powerful tools, make the targets of fast evaluation of adsorbents and evolving materials for CO_2 capture and CH_4 storage feasible. This review provides an overview of the recent computational efforts on such related topics and discusses the further development in this field.

An optimized micro-plate assay for high-throughput screening of recombinant Pichia pastoris strains

Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.

High-throughput Screening of the Enantioselectivity of Hyperthermophilic Mutant Esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

Development of a New High-throughput Screening Model for Human High Density Lipoprotein Receptor (CLA-1) Agonists

To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

A high-throughput screening method for breeding Aspergillus niger with 12C6+ ion beam-improved cellulase

In this study,a high-throughput screening method was established through the 24-square deep-well microliter plate(MTP) fermentation and micro-plate detection for large-scale screening of the mutants.It was suitable for screening a large number of mutants and improving the breeding efficiency after heavy-ion beam irradiation.Seventeen strains showed higher cellulase activity compared with the initial strain after the screening of plate and MTP fermentation.The filter paper activity and β-glucosidase activity of Aspergillus niger H11201 had increased 38.74 and 63.23%separately compared with A.niger H11 by shaking flask fermentation,and it was genetically stable after being passaged to nine generations.The results indicate that the high-throughput screening method can be used for the quick breeding of A.niger with high cellulase activity.

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

Suppression of cellular O-linkedβ-N-acetylglucosaminylation(O-Glc NAcylation)can repress proliferation and migration of various cancer cells,which opens a new avenue for cancer therapy.Based on the regulation of insulin gene transcription,we designed a cell-based fluorescent reporter capable of sensing cellular O-Glc NAcylation in HEK293 T cells.The fluorescent reporter mainly consists of a reporter(green fluorescent protein(GFP)),an internal reference(red fluorescent protein),and an operator(neuronal differentiation 1),which serves as a"sweet switch"to control GFP expression in response to cellular OGlc NAcylation changes.The fluorescent reporter can efficiently sense reduced levels of cellular OGlc NAcylation in several cell lines.Using the fluorescent reporter,we screened 120 natural products and obtained one compound,sesamin,which could markedly inhibit protein O-Glc NAcylation in He La and human colorectal carcinoma-116 cells and repress their migration in vitro.Altogether,the present study demonstrated the development of a novel strategy for anti-tumor drug screening,as well as for conducting gene transcription studies.

High-Throughput Screening of Nanoparticle-Stabilizing Ligands:Application to Preparing Antimicrobial Curcumin Nanoparticles by Antisolvent Precipitation

Water-dispersible curcumin nanoparticles were prepared by bottom-up antisolvent precipitation approach. A new high-throughput screening technique was developed for selecting appropriate ligands stabilizing the nanoparticles in aqueous medium and improving their performance. The initial set of twenty-eight potential stabilizing ligands was evaluated based on their capacity to improve curcumin dispersibility in aqueous medium. The performance of four promising ligands(amino acid proline, polyphenol tannic acid, polycation Polyquaternium 10, and neutral polymer polyvinylpyrrolidone) was tested in ultrasound-aided antisolvent precipitation trials. Using the selected stabilizing ligands diminished the average particle size from ca. 1,200 to 170–230 nm, reduced their dispersity, improved stability, and allowed reaching curcumin concentration of up to 1.4 m M in aqueous medium. Storage stability of the aqueous nanodispersions varied from 2 days to 2 weeks, depending on stabilizing ligand. Studying the effects of ionic strength and pH on size and f-potential of the particles suggested that electrostatic forces and hydrophobic interactions could be the major factors affecting their stability. The ligand-protected nanoparticles showed minimal inhibitory concentration of 400 or500 μM toward Escherichia coli. We suggest that the presented screening approach may be useful for preparing nanoparticles of various poorly water-soluble bioactive materials.

Application and prospects of high-throughput screening for in vitro neurogenesis

Over the past few decades,high-throughput screening(HTS)has made great contributions to new drug discovery.HTS technology is equipped with higher throughput,minimized platforms,more automated and computerized operating systems,more efficient and sensitive detection devices,and rapid data processing systems.At the same time,in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes.However,challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional(3D)in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions.Here,we review the applications of HTS technologies on in vitro neurogenesis,especially aiming at identifying the essential genes,chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids.We also discuss the developmental tendency of HTS technology,e.g.,so-called next-generation screening,which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.

Computational high-throughput screening and in vitro approaches identify CB-006-3;A novel PI3K-BRAF^(V600E) dual targeted inhibitor against melanoma

Malignant melanoma is characterized by both genetic and molecular alterations that activate phosphoinositide 3-kinase(PI3K),and RAS/BRAF pathways.In this work,through diversity-based high-throughput virtual screening we identified a lead molecule that selectively targets PI3K and BRAF^(V600E) kinases.Computational screening,Molecular dynamics simulation and MMPBSA calculations were performed.PI3K and BRAF^(V600E) kinase inhibition was done.A375 and G-361 cells were used for in vitro cellular analysis to determine antiproliferative effects,annexin V binding,nuclear fragmentation and cell cycle analysis.Computational screening of small molecules indicates compound CB-006-3 selectively targets PI3KCG(gamma subunit),PI3KCD(delta subunit)and BRAF^(V600E).Molecular dynamics simulation and MMPBSA bases binding free energy calculations predict a stable binding of CB-006-3 to the active sites of PI3K and BRAF^(V600E).The compound effectively inhibited PI3KCG,PI3KCD and BRAF^(V600E)kinases with respective IC50 values of 75.80,160.10 and 70.84 nM.CB-006-3 controlled the proliferation of A375 and G-361 cells with GI50 values of 223.3 and 143.6 nM,respectively.A dose dependent increase in apoptotic cell population and sub G0/G1 phase of cell cycle were also observed with the compound treatment in addition to observed nuclear fragmentation in these cells.Furthermore,CB-006-3 inhibited BRAF^(V600E),PI3KCD and PI3KCG in both melanoma cells.Collectively,based on the computational modeling and in vitro validations,we propose CB-006-3 as a lead candidate for selectively targeting PI3K and mutant BRAF^(V600E) to inhibit melanoma cell proliferation.Further experimental validations,including pharmacokinetic evaluations in mouse models will identify the druggability of the proposed lead candidate for further development as a therapeutic agent for treating melanoma.

Screening and Identification of Myxobacterias with Antifungal Activity

[ Objective] The paper was to screen myxobacteria with antifungal activity, and identify its species. [ Method] 237 myxobacteria were isolated and pu- rified from 200 copies of soil samples collected from 23 provinces in China. The strains were fermented in four different media, and the fermentation products were extracted by methanol to obtain metabolites. With Candida albicans as the model, the secondary metabolites were carried out high-throughput screening. [ Result ] A strain of myxohacteria numbered ZJ2 was obtained after secondary screening, its secondary metabolites had activities against G. albicans. 16S rDNA cloning and sequencing results showed that ZJ2 was Myxococcus fidvus. [ Conclusion ] The secondary metabolites of ZJ2 strain had bioactive substances with significant inhibi- tion effect against the growth of C. albicans, which had potential pharmaceutical research and development value.