High-throughput Screening Process Research Articles

Deep-PK: deep learning for small molecule pharmacokinetic and toxicity prediction.

Evaluating pharmacokinetic properties of small molecules is considered a key feature in most drug development and high-throughput screening processes. Generally, pharmacokinetics, which represent the fate of drugs in the human body, are described from four perspectives: absorption, distribution, metabolism and excretion-all of which are closely related to a fifth perspective, toxicity (ADMET). Since obtaining ADMET data from in vitro,in vivo or pre-clinical stages is time consuming and expensive, many efforts have been made to predict ADMET properties via computational approaches. However, the majority of available methods are limited in their ability to provide pharmacokinetics and toxicity for diverse targets, ensure good overall accuracy, and offer ease of use, interpretability and extensibility for further optimizations. Here, we introduce Deep-PK, a deep learning-based pharmacokineticand toxicity prediction, analysis and optimization platform. We applied graph neural networks and graph-based signatures as a graph-level feature to yield the best predictive performance across 73 endpoints, including 64 ADMET and 9 general properties. With these powerful models, Deep-PK supports molecular optimization and interpretation, aiding users in optimizing and understanding pharmacokinetics and toxicity for given input molecules. The Deep-PK is freely available athttps://biosig.lab.uq.edu.au/deeppk/.

Asymmetric synthesis of enantiopure tetracyclic dispirooxindolopyrrolidine-piperidones via microwave-assisted multicomponent reaction: Crystallographic analysis, antimicrobial activity and in silico studies

In an attempt towards the development of new antimicrobials, we synthesized by microwave-assisted multicomponent reaction (MCR) of enantiopure (E,E)-3,5-bisarylidene-N-[(S)-(-)-methylbenzyl]-4-piperidones, isatin and α-amino esters a series of optically active tetracyclic dispirooxindolopyrrolidine-piperidones. The beneficial and promising features of this protocol such as simple operational procedure, short reaction time (10 min), and high product yields (up to 97 %) make it an efficient eco-friendly approach offering a powerful mean to expand the structural diversity of spirooxindoles. This approach appears hugely advantageous for high-throughput screening processes in drug discovery research. In addition, crystallographic parameters and inter- and intramolecular interactions occurring in spiropyrrolidine-fused piperidone derivative 4g were examined through single-crystal XRD analysis allowing to determine the absolute configuration of the enantiopure compound. The preliminary biological assessment of their antimicrobial activity indicates that most of the screened products display higher activity than the standard reference drugs Ampicillin and Griseofulvin. Some of them exhibited good to moderate activity against both bacteria and fungi. In addition, in silico molecular docking and predictive ADMET studies for the most active spirocompounds were carried out. Molecular docking confirmed the binding efficacy of candidates 4c and 4l through low score energy values and the establishment of diverse bonding interactions with the active site residues. Finally, the ADMET profiling of the most active spiroheterocycles proved their remarkable drug-like and pharmacokinetic properties.

Accelerated Screening of Ternary Chalcogenides for Potential Photovoltaic Applications.

Chalcogenides, which refer to chalcogen anions, have attracted considerable attention in multiple fields of applications, such as optoelectronics, thermoelectrics, transparent contacts, and thin-film transistors. In comparison to oxide counterparts, chalcogenides have demonstrated higher mobility and p-type dopability, owing to larger orbital overlaps between metal-X covalent chemical bondings and higher-energy valence bands derived by p-orbitals. Despite the potential of chalcogenides, the number of successfully synthesized compounds remains relatively low compared to that of oxides, suggesting the presence of numerous unexplored chalcogenides with fascinating physical characteristics. In this study, we implemented a systematic high-throughput screening process combined with first-principles calculations on ternary chalcogenides using 34 crystal structure prototypes. We generated a computational material database containing over 400,000 compounds by exploiting the ion-substitution approach at different atomic sites with elements in the periodic table. The thermodynamic stabilities of the candidates were validated using the chalcogenides included in the Open Quantum Materials Database. Moreover, we trained a model based on crystal graph convolutional neural networks to predict the thermodynamic stability of novel materials. Furthermore, we theoretically evaluated the electronic structures of the stable candidates using accurate hybrid functionals. A series of in-depth characteristics, including the carrier effective masses, electronic configuration, and photovoltaic conversion efficiency, was also investigated. Our work provides useful guidance for further experimental research in the synthesis and characterization of such chalcogenides as promising candidates, as well as charting the stability and optoelectronic performance of ternary chalcogenides.

A semi-rational mutagenesis approach for improved substrate activity of microbial transglutaminase.

A higher specific activity of microbial transglutaminase (mTGase) is desirable for a broad range of applications ranging from food industry to biotechnology. Three-dimensional docking simulation of mTGase revealed that residues V65, W69, and Y75 were critical for substrate recognition. A semi-rational mutagenesis approach was applied to each residue to generate three separate mini mutant libraries. A high-throughput screening process identified five mutants that demonstrated improved specific activities than the wild type (WT) mTGase were isolated from the Y75 mini mutant library. Mutant Y75L showed approximately 60% increment in specific activity and improved substrate specificity. Conjugation of two heterologous single-chain fragment variable clones to generate a diabody with mutant Y75L was successfully performed and validated. This work demonstrates the successful application of semi-rational mutagenesis coupled with a high-throughput screening approach to identify mTGase mutants with improved specific activities and specificities which are beneficial for protein-protein conjugation.

Construction of a QSAR Model Based on Flavonoids and Screening of Natural Pancreatic Lipase Inhibitors.

Pancreatic lipase (PL) is a key hydrolase in lipid metabolism. Inhibition of PL activity can intervene in obesity, a global sub-health disease. The natural product is considered a good alternative to chemically synthesized drugs due to its advantages, such as low side effects. However, traditional experimental screening methods are labor-intensive and cost-consuming, and there is an urgent need to develop high-throughput screening methods for the discovery of anti-PL natural products. In this study, a high-throughput virtual screening process for anti-PL natural products is provided. Firstly, a predictable anti-PL natural product QSAR model (R2train = 0.9444, R2test = 0.8962) were developed using the artificial intelligence drug design software MolAIcal based on genetic algorithms and their conformational relationships. 1068 highly similar (FS > 0.8) natural products were rapidly enriched based on the structure-activity similarity principle, combined with the QSAR model and the ADMET model, for rapid prediction of a total of five potentially efficient anti-PL natural products (IC50pre < 2 μM). Subsequently, molecular docking, molecular dynamics simulation, and MMGBSA free energy calculation were performed to not only reveal the interaction of candidate novel natural products with the amino acid residues of PL but also to validate the stability of these novel natural compounds bound to PL. In conclusion, this study greatly simplifies the screening and discovery of anti-PL natural products and accelerates the development of novel anti-obesity functional foods.

Abstract P2160: High-throughput Imaging Of Cardiomyocytes Using Biomimetic Tools Help Identify Therapeutic Targets

Cardiovascular disease is a leading cause of death and continues to require novel forms of therapies. Mutations in sarcomeres, the basic contractile units of cardiac muscle, causing disarray and loss of function are causal in many genetic cardiomyopathies. Here, we model sarcomeric disarray in vitro by culturing human induced pluripotent stem cell-derived cardiomyocytes (human iPSC-CMs) on tissue culture plates with biomimetic patterned grooves designed to simulate extracellular matrix. This culture system models the natural structure and phenotypic development of human physiology. Next, we created an Adeno-associated virus open reading frame (AAV-ORF) library of ~100 heart failure associated genes of interest (GOI) identified using publicly available human genetic data. We developed a high throughput screening (HTS) process to test the effects of overexpression of these GOIs on sarcomere morphology. Images of human iPSC-CMs treated with overexpression libraries were run through a custom MATLAB HTS image processing script named ‘Tamarack’, developed for quantifying human iPSC-CM sarcomere morphology. Tamarack enables quantification of sarcomere count, length, and orientation. Analysis of sarcomeres from the Tamarack code in the biomimetic plates allows for enhanced quantification of sarcomere structures, in particular, alignment, a metric missing from standard 2D culture plates. We found that treating cells with siRBM20 induced a sarcomere misalignment phenotype which we looked to rescue with overexpression of our GOI. Genes that increased alignment in an RBM20 background were considered top hits and moved to further analysis.

A high throughput screening process and quick isolation of novel lignin-degrading microbes from large number of natural biomasses

High throughput screening approaches can significantly speed up the identification of novel enzymes from natural microbial consortiums. A two-step high throughput screening process was proposed and explored to screen lignin-degrading microorganisms. By employing this modified culture enrichment method and screening based on enzyme activity, a total of 82 bacterial and 46 fungal strains were isolated from fifty decayed wood samples (100 liquid cultures) collected from the banks of the Ottawa River in Canada. Among them, ten bacterial and five fungal strains were selected and identified based on their high laccase activities by 16S rDNA and ITS gene sequencing, respectively. The study identified bacterial strains from various genera including Serratia, Enterobacter, Raoultella, and Bacillus, along with fungal counterparts including Mucor, Trametes, Conifera and Aspergillus. Moreover, Aspergillus sydowii (AORF21), Mucor sp. (AORF43), Trametes versicolor (AORF3) and Enterobacter sp. (AORB55) exhibited xylanase and β- glucanase activities in addition to laccase production. The proposed approach allowed for the quick identification of promising consortia and enhanced the chance of isolating desired strains based on desired enzyme activities. This method is not limited to lignocellulose and lignin-degrading microorganisms but can be applied to identify novel microbial strains and enzymes from different natural samples.

A high-throughput molecular dynamics screening (HTMDS) approach to the design of novel cyclopeptide inhibitors of ATAD2B based on the non-canonical combinatorial library

Cyclic peptides (CPs) are a promising class of drugs because of their high biological activity and specificity. However, the design of CP remains challenging due to their conformational flexibility and difficulties in designing stable binding conformation. Herein, we present a high-throughput MD screening (HTMDS) process for the iterative design of stable CP binders with a combinatorial CP library composed of canonical and non-canonical amino acids. As a proof of concept, we apply our methods to design CP inhibitors for the bromodomain (BrD) of ATAD2B. 698,800 CP candidates with a total of 25,570 ns MD simulations were performed to study the protein-ligand binding interactions. The binding free energies (ΔGbind) estimated by MM/PBSA approach for eight lead CP designs were found to be low. CP-1st.43 was the best CP candidate with an estimated ΔGbind of −28.48 kcal/mol when compared to the standard inhibitor C-38 which has been experimentally validated and shown to exhibit ΔGbind of −17.11 kcal/mol. The major contribution of binding sites for BrD of ATAD2B involved the hydrogen-bonding anchor within the Aly-binding pocket, salt bridging, and hydrogen-bonding mediated stabilization of the ZA loop and BC loop, and the complementary Van der Waals attraction. Our methods demonstrate encouraging results by yielding conformationally stable and high-potential CP binders that should have potential applicability in future CP drug development. Communicated by Ramaswamy H. Sarma

Enhancing the interfacial stability of all-solid-state high-energy sodium-ion batteries by coating materials: First principles calculations

All-solid-state batteries are promising next-generation batteries owing to their enhanced safety and high energy density. Recently, all-solid-state sodium-ion batteries have broadly investigated owing to low price and abundance of sodium. Various types of solid electrolytes (SEs) exhibiting high ionic conductivities have been investigated for all-solid-state Na-ion batteries. However, a few studies have been reported for the interfacial stability of sodium SEs against high-energy cathode materials. In this study, we systematically investigated the interfacial stability between ten sodium superionic conductors and 16 high-energy oxide cathode materials. The calculation results indicated that sodium sulfide and selenide SEs generally exhibit chemical incompatibility with cathode materials, suggesting that interfacial coating should be employed to alleviate the interfacial reaction. We utilized high-throughput screening process to identify promising coating materials between sodium SEs and cathode materials. Eight compounds from 7,398Na-containing compounds were screened as promising coating materials, which considerably reduced the chemical reactivity at the interface. The findings of this study can provide design strategies for the all-solid-state sodium-ion batteries.

Quantitative Phase Field Modeling of Li Dendrite Growth

Modeling and understanding dendrite growth on lithium metal anodes during charging is vital for improving battery safety. The phase field method has become an increasingly popular tool to achieve this due to its flexibility in handling complex morphologies and coupled physical processes. However, quantitative phase field models under realistic electrochemical conditions are rare. In this work, we derive a thermodynamically consistent phase field model in the grand potential framework. The design of this model emphasizes quantitative modeling of electrodeposition with experimental loading conditions and realistic length and time scales. First, electrostatic potentials in both the electrode and the electrolyte are considered to account for the interfacial potential difference. Second, the electrochemical driving force can be large even with an applied voltage of a few hundred millivolts; therefore, the grid size usually must be as small as a few nanometers to maintain a stable phase field profile. This hinders the modeling of system sizes of practical interest. To solve this problem, a mapping approach is introduced to maintain stability with moderate grid sizes and realistic applied voltages. Third, we thoroughly compare the phase field model with results from a sharp interface model for various cases that permit analytical solutions. Numerical studies demonstrate that the proposed model can quantitatively reproduce the sharp interface results. Moreover, lithium has highly anisotropic interfacial energy, which leads to corners on the equilibrium electrode-electrolyte interface shape. A convexification method is used to remove the ill-posedness due to this strong anisotropy. Finally, a parallel solver is developed to handle large systems. The overall framework is demonstrated by studying lithium dendrite morphologies under various conditions. In future work, the model can be used as a computational tool for the high-throughput screening process in battery design.

Enhancing protease activity of Bacillus subtilis using UV-laser random mutagenesis and high-throughput screening

Increasing the peptide level of fermented soybean meal helps improve its nutritional value, so it is necessary to screen strains with high protease activity. In this study, UV-laser random mutagenesis was employed to treat Bacillus subtilis for the selection of high protease producers. A high-throughput screening (HTS) processed aided by a microtiter plate (MTP) was established to enhance the efficiency of screening. Five mutagenesis methods were employed to investigate the mutagenesis effect, among which the positive mutation rate was the highest in laser UV sequential mutation (LA-UV). Two mutants, UV-1–84 and LA-UV-1–11, were re-screened from 32 positive mutants, exhibiting excellent genetic stability and protease activity. Besides, fermented soybean meal test showed that peptide content of SBM fermented by mutants UV-1–84 and LA-UV-1–11 increased by 13.66% and 12.91% respectively, compared with the wild-type strain. The HTS process established in this study could be easily transferred to other similar high-yield strains.

PD43-09 PHENOTYPICAL SCREENING ONMETASTATIC PRCC-TFE3 FUSIONTRANSLOCATION RENAL CELL CARCINOMA ORGANOIDS REVEALSPOTENTIAL THERAPEUTIC AGENTS

You have accessJournal of UrologyCME1 May 2022PD43-09 PHENOTYPICAL SCREENING ONMETASTATIC PRCC-TFE3 FUSIONTRANSLOCATION RENAL CELL CARCINOMA ORGANOIDS REVEALSPOTENTIAL THERAPEUTIC AGENTS Chuanzhen Cao, Xiaomei Lan, Weixing Jiang, Lei Guo, Xingang Bi, Shan Zheng, Aiping Zhou, Zhijian Sun, and Jianzhong Shou Chuanzhen CaoChuanzhen Cao More articles by this author , Xiaomei LanXiaomei Lan More articles by this author , Weixing JiangWeixing Jiang More articles by this author , Lei GuoLei Guo More articles by this author , Xingang BiXingang Bi More articles by this author , Shan ZhengShan Zheng More articles by this author , Aiping ZhouAiping Zhou More articles by this author , Zhijian SunZhijian Sun More articles by this author , and Jianzhong ShouJianzhong Shou More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002604.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Translocation renal cell carcinoma (tRCC) is a subtypethat occurred predominantly in children and young. Metastatic tRCC occurred in young is more aggressive and there are still no effective therapies. Organoidscan mimic original tissues then adopt to high-throughput screening (HTS). We aim to utilize patient-derived organoid and HTS to screen repurposing drugs for metastatic PRCC-TFE3 fusion tRCC. METHODS: Tumor tissues were obtained from treatment-naïve metastatic tRCC patients who underwent surgeries.Pathology, fluorescence in situ hybridization (FISH) confirmed the PRCC-TFE3 fusion tRCC. Organoids were cultured and verified byFISH and RNA-seq.HTS was performed to seek promising drugs and potential mechanisms were analyzed by RNA-seq. RESULTS: We successfully established a metastatic PRCC-TFE3 fusion tRCC organoid of a common fusion subtypeand characterization was verified by histopathology, FISH, and RNA-seq. HTS assay was developed and the robustness was confirmed. A compound library of 1816 drugs was screened. Eventually, axitinib, crizotinib, and JQ-1were selected for further validation, which could induce cell cycle arrest and apoptosis. RNA-seq analyses of post-treatment organoids indicated crizotinib induced significant autophagy-related gene alternations which were consistent with tRCC potential pathogenesis. CONCLUSIONS: We established and validated the organoid cultures from tissues starting from a metastatic PRCC-TFE3 fusion tRCC patient and achieved the HTS process for the first time. Crizotinib might be the targeted therapy worthy of exploring in clinic for metastatic PRCC-TFE3 fusion tRCC.Such organoid and HTS assay may represent a promising model system in translational research assisting clinical strategies. Source of Funding: This work was supported by theNational Science and Technology Major Project of China (ID Number: 2016ZX09101094) and the National Natural Science Foundation of China (ID Number: 82072837) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e705 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chuanzhen Cao More articles by this author Xiaomei Lan More articles by this author Weixing Jiang More articles by this author Lei Guo More articles by this author Xingang Bi More articles by this author Shan Zheng More articles by this author Aiping Zhou More articles by this author Zhijian Sun More articles by this author Jianzhong Shou More articles by this author Expand All Advertisement PDF DownloadLoading ...

A system for multiplexed selection of aptamers with exquisite specificity without counterselection

Aptamers have proven to be valuable tools for the detection of small molecules due to their remarkable ability to specifically discriminate between structurally similar molecules. Most aptamer selection efforts have relied on counterselection to eliminate aptamers that exhibit unwanted cross-reactivity to interferents or structurally similar relatives to the target of interest. However, because the affinity and specificity characteristics of an aptamer library are fundamentally unknowable a priori, it is not possible to determine the optimal counterselection parameters. As a result, counterselection experiments require trial-and-error approaches that are inherently inefficient and may not result in aptamers with the best combination of affinity and specificity. In this work, we describe a high-throughput screening process for generating high-specificity aptamers to multiple targets in parallel while also eliminating the need for counterselection. We employ a platform based on a modified benchtop sequencer to conduct a massively parallel aptamer screening process that enables the selection of highly specific aptamers against multiple structurally similar molecules in a single experiment, without any counterselection. As a demonstration, we have selected aptamers with high affinity and exquisite specificity for three structurally similar kynurenine metabolites that differ by a single hydroxyl group in a single selection experiment. This process can easily be adapted to other small-molecule analytes and should greatly accelerate the development of aptamer reagents that achieve exquisite specificity for their target analytes.

Phenotypical screening on metastatic PRCC-TFE3 fusion translocation renal cell carcinoma organoids reveals potential therapeutic agents

PurposeTranslocation renal cell carcinoma (tRCC) is a subtype that occurs predominantly in children and young individuals. Metastatic tRCC occurring in young patients is more aggressive than that occurring in older patients, and there are still no effective therapies. Organoids can mimic original tissues and be assessed by high-throughput screening (HTS). We aimed to utilize patient-derived organoids and HTS to screen drugs that can be repurposed for metastatic tRCC with PRCC-TFE3 fusion.MethodsTumor tissues were obtained from treatment-naïve metastatic tRCC patients who underwent surgery. Histopathology and fluorescence in situ hybridization (FISH) confirmed the tRCC. Organoids derived from the dissected tissues were cultured and verified by FISH and RNA-seq. HTS was performed to seek promising drugs, and potential mechanisms were explored by RNA-seq and cell-based studies.ResultsWe successfully established a metastatic tRCC organoid with PRCC-TFE3 fusion, a common fusion subtype, and its characteristics were verified by histopathology, FISH, and RNA-seq. An HTS assay was developed, and the robustness was confirmed. A compound library of 1816 drugs was screened. Eventually, axitinib, crizotinib, and JQ-1 were selected for further validation and were found to induce cell cycle arrest and apoptosis. RNA-seq analyses of posttreatment organoids indicated that crizotinib induced significant changes in autophagy-related genes, consistent with the potential pathogenesis of tRCC.ConclusionsWe established and validated organoids derived from tissues dissected from a patient with metastatic tRCC with PRCC-TFE3 fusion and achieved the HTS process for the first time. Crizotinib might be a targeted therapy worthy of exploration in the clinic for metastatic tRCC with PRCC-TFE3 fusion. Such organoid and HTS assays may represent a promising model system in translational research assisting in the development of clinical strategies.

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products.Key points• A protein expression vector using double-antibiotic resistance genes was designed• Double antibiotic selection causes fewer colonies with more positive for phenotype• Coupling the new vector with FACS improves microalgal screening efficiency > 60%

Isoreticular chemistry within metal–organic frameworks for gas storage and separation

• The application of isoreticular chemistry in MOF structure design, including microstructure design, pore size tailoring, secondary building unit modification, post-synthesis modification, and cooperative regulation are summarized and highlight. • The adsorption/separation function-oriented MOF structure design and high-throughput screening process are summarized. Based on isoreticular chemistry design and optimized high-performance MOF adsorbents and membranes, we are committed to exploring an energy-intensive and environmentally friendly alternative separation route, and strive to achieve efficient separation of hydrogen, carbon dioxide, natural gas, and petroleum-based platform compounds. Precise control of the pore size and environment of metal–organic framework (MOF) is a necessary condition for achieving high performance of gas adsorption and separation. After nearly two decades of development, the synthesis of MOF materials has gradually evolved from exploration and trial to precise-design, including function-oriented microstructure design and optimization, pore size tailoring, and secondary building unit (SBU) modification. The unique pore environments of MOF materials enable their advantages in gas adsorption and separation applications. In addition, the introduction of isoreticular chemistry within MOFs (with the same framework structure and different chemical components) provides opportunities for improving gas adsorption and separation performance. Isoreticular chemistry gives MOFs more functions to promote specific binding or sieving with gas molecules. Furthermore, MOF-based adsorbents and separation membranes exhibit superior separation performance in many industrial gas purification processes. In this review, we summarized and highlight the application of isoreticular chemistry in MOF structure design, including microstructure design, pore size tailoring, SBU modification, post-synthesis modification, and cooperative regulation. The gas adsorption and separation performances are improved through pore size and environment optimization. In addition, we also summarized the adsorption/separation function-oriented MOF structure design and high-throughput screening process. Based on isoreticular chemistry design and optimized high-performance MOF adsorbents and separation membranes, an energy-intensive and environmentally friendly alternative separation route is explored to achieve efficient separation of hydrogen, carbon dioxide, natural gas, and petroleum-based compounds. Finally, we provided an outlook based on prospect developments of isoreticular chemistry within MOFs for gas storage and separation.

NanoLuc (NLuc) complementation assay elucidates role of specific G‐proteins in GPR88 signaling

GPR88 is an orphan G protein-coupled receptor (GPCR) expressed at high levels in the striatum and is a target for the development of therapeutics to treat multiple neuropsychiatric disorders. While a few small-molecule synthetic agonists have been reported, no synthetic antagonists or endogenous ligands have been identified. Typical 2nd messenger, cell-based assays for agonist and antagonist identification and signaling pathway elucidation rely on complex downstream events, with interpretation of results complicated from effects of transfected receptor overexpression and interference from endogenous receptors and proteins. The NLuc complementation assay, based on an engineered luciferase from the deep-sea shrimp, Oplophorus gracilirostris, can monitor receptor-G protein interaction in live cells and profile compounds according to the G proteins they activate. The synthetic agonists, 2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane carboxylic acid], and 2-OMPP [(2R)-N-(2-hydroxy-1-(4-((2-methylpentyl)oxy)phenyl)ethyl)-2-phenylpropanamide] are suspected to activate GPR88 via a Gαi-coupled signaling pathway. In this study we show the activation of GPR88 by 2-PCCA and 2-OMPP inhibited isoproterenol induced cAMP production in transiently transfected GPR88 HEK-293T cells with relative EC50 values of 250 nM and 1.0 μM, respectively, consistent with prior reports. The EC50 value for PCCA in HEK-293T/GPR88 transients is five times that in RH-7777 cells with COS-7 cells showing no cAMP response. These results highlight the importance of the cellular background in engineered systems. Additionally, the NLuc complementation assay shows PCCA receptor binding triggers activation of several G-proteins including Gαi1(EC50= 760 nM), Gαi0 (EC50=3.3μM) and GαS (EC50=4.5 nM) indicating the effects of varying G-protein expression in different cell lines. In combination with our computational endeavors, 2nd messenger and the NLuc complementation assays are valuable tools in our research as we develop high-throughput screening processes and probe signaling pathways in search of GPCR based therapeutics.

Semi-quantitative activity assays for high-throughput screening of higher activity gamma glutamyl transferase and enzyme immobilization to efficiently synthesize L-theanine

The bio-production of theanine is currently of significant interest due to its wide applications in food and healthcare products. Gamma glutamyl transferase (GGT) has been widely applied in L-theanine synthesis, but L-theanine yields remain prohibitively low for commercial production. In this study, a robust high-throughput screening process for isolating GGT mutants was developed through a combination of error-prone PCR techniques and a colorimetric reaction. The co-expression of PrsA lipoprotein enhances the secretion of GGT, thus GGT could be obtained quickly and easily without crushing cells. Random mutations on ggt genes were introduced by using error-prone PCR kits to build a large mutant library. A colorless compound generated by the reaction between NH4+ (released from L-theanine synthesis) and OPA was measured quantitatively by UV/visible spectroscopy when mixed with TCA and DMSO. Approximately 30 positive clones with improved color formation on the 96-well plates were identified, and mutants T413P and T463S with more than by 30 % higher transpeptidation activity versus the original GGT were isolated. To improve the operational stability and economical use, mutant GGT was immobilized on a prepared oxidized cellulose nanofiber membrane. The remaining activity of immobilized GGT was 88 % versus 72 % of free enzyme over 15 h. A fed-batch conversion was performed with the immobilized GGT, and over 70 g/L L-theanine could be accumulated within 18 h after feeding twice. Versus other studies, this is one of the best L-theanine synthesis systems using immobilized GGT.

(Invited) Density Functional Theory Modeling - Machine Learning Approach for Redox Potential of Organic Electrode Materials

In this talk, we present the first-principles density functional theory modeling of redox properties of various carbon materials, including quinone derivatives, dopamine, and DNA. First, we discuss the contribution of electron affinity of molecules and solvation to redox potential from our density functional theory (DFT) calculations. Second, we discuss the relationship of redox potential with the design of molecular structure. It is found that aromaticity and electron-withdrawing functional groups can be used to tune the redox potential. Especially, the variation of redox potential as a function of Li association is discussed. The conclusion is that we can tune up the redox potential by designing the detailed molecular structure. Next, all these results are used to build up machine learning. We discuss our artificial neural network and other machine learning algorithms regarding its validation and predictability. Through this study, the redox characteristics of carbon materials are systematically investigated using the first-principles computational modeling method, and then utilized via machine learning methods for high-throughput screening process.

Novel Targets for Antimicrobials.

Antimicrobial resistance (AMR) is the phenomenon developed by microorganism on exposure to antimicrobial agents, making them unresponsive. Development of microbial confrontation is a severe rising risk to global community well-being as treatment in addition, management of such resistant microbial infections is difficult and challenging. The situation requires action across all government sectors and society. The change in the molecular target on which antimicrobial drugs act is one of the key mechanisms behind AMR. One of the approaches to battle with AMR can be exploring newer molecular targets in microbes and discovering new molecules accordingly. There are various examples of novel targets such as biomolecules involving in biosynthesis of cell wall, biosynthesis of aromatic amino acid, cell disunion, biosynthesis of fatty acid, and isoprenoid biosynthesis and tRNA synthetases. Fatty acid biosynthesis (FAB) and their enzymes among all the above is the more appealing target for the advancement of new antimicrobial agents. Number of promising inhibitors have been developed for bacterial fatty acid synthesis (FAS) and also few of them are clinically used. Some of these potential inhibitors are found to be used in development of new antibacterial as a lead compound and have been discovered from high throughput screening processes like Platencimycin and their analogue, Platencin. The review majorly encompasses bacterial FAB in type II FAS system and potential inhibitors with respective targets of novel antibacterial.