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Metal-organic frameworks (MOFs) are crystalline porous materials with excellent gas storage capacities and unique chemical tunability. Their application as new materials to uptake and degrade chemical warfare agents has been the topic of intense study over the last half-decade. Work by others proved that Zr-MOFs and polymer composites made from them are contenders for the next generation of personal protective equipment (PPE) for combatting nerve agents.In an attempt to speed up discovery and understanding of MOF catalysts for nerve agent degradation, a high-throughput screening (HTS) assay was developed for a common nerve agent simulant. Chapter 2 outlines the development of this assay as well as the curation and screening results from the library of 93 MOFs and related materials.Successful development and validation of the HTS assay led to targeted attempts to apply this method for both a wider array of MOFs as well as MOF-polymer composites. Chapter 3 uses the speed of HTS to investigate two groups of closely related MOFs that result in a wide range of catalytic activities. This chapter also adapts the HTS assay to analyze composites and again finds that small differences in materials changes the outcome of catalyst performance.Chapter 4 describes the development of a new HTS assay with another nerve agent simulant that can be directly compared to results of the assay developed in Chapter 2. This new assay expands understanding of the degradation attempts by MOF catalysts to date. In addition, this new HTS method was compared to a medium-throughput method for monitoring breakdown of nerve agent VX and was confirmed by literature assay using 31P NMR.MOF catalysts design was paired with MOF-polymer composite design, when a new strong, ductile hybrid was developed in Chapter 5. Catalytically active MOFs were combined with a spandex polymer to target composites for durable PPE. The HTS assay was used to show catalytic ability of these composites and three processing methods demonstrated the versatility of these MOF-polymer composites.

Protein-protein interactions (PPIs) are ubiquitous in Nature and found in all domains of life. They mediate signal transduction, muscle contraction, electron transport and metabolic pathway processing, among many other processes, and the molecular underpinnings of PPIs remain a rich source of scientific exploration. One model system used to study PPIs is fatty acid biosynthesis (FAS) from Escherichia coli. In E. coli FAS each enzyme of the pathway is a discrete unit to which cargo is delivered by a central acyl carrier protein (EcACP). EcACP tethers the growing acyl chain, sequesters the chain within its alpha helical core to protect it from hydrolysis and cross reactivity, finds the correct partner enzyme, delivers the cargo to the correct partner enzyme and then finds the next partner enzyme in the cycle, all while navigating the crowded milieu of the cytoplasm. During E. coli FAS, EcACP engages in nine different PPIs, and has been implicated in at least 23 additional PPIs with enzymes and proteins from other metabolic pathways in E. coli alone. This wide range of PPIs somehow allow EcACP to deliver substrates to partner enzymes with exquisite selectivity and specificity.While numerous biophysical and structural tools such as isothermal titration calorimetry or X-ray crystallography exist to characterize PPIs, progress in the field has been hindered by a lack of universal, or rapid screen. This dissertation focuses on the development of four different fluorescent-based assays for the rapid visualization of PPIs between EcACP and its numerous partner enzymes, and for the discovery of new carrier protein interactions in previously uncultured marine bacteria. First, the utility of a 4-dimethylnaphthalene (4-DMN) pantetheine probe to monitor thirteen different EcACP PPIs was expanded. 4-DMN-pantetheine was appended to EcACP to generate 4-DMN-EcACP and it was shown that when a partner enzyme interacted with 4-DMN-EcACP, the 4-DMN cargo from EcACP was chain flipped into the active site of the partner enzyme, which led to an increase in fluorescence intensity. 4-DMN-EcACP was used to visualize PPIs between EcACP and enzymes from FAS, biotin and Lipid A biosynthesis, iron-sulfur cluster formation and chromosome trafficking to demonstrate the wide applicability of this fluorescent tool. It was also shown that this fluorescent tool could be used to detect the presence of active site inhibitors in partner enzymes.Next, in silico screening methods were used to identify a potential inhibitor of the PPI between EcACP and a dehydratase from E. coli FAS, FabA. Following identification of a lead compound, suramin, the inhibitory effect of suramin was tested in vitro using the previously developed solvatochromic assay, and the inhibition of suramin was confirmed using a secondary, crosslinking based assay. This work provided the basis for a major R01 grant to the NIH and demonstrated how detailed structural knowledge of these complexes can guide antibiotic discovery efforts and the development of a high-throughput screening program to be undertaken with collaborators at Sanford-Burnham-Prebys. The utility of 4-DMN-EcACP was also demonstrated for mapping residues on the surface of two partner enzymes from E. coli FAS, FabF and FabI. Previous structural methods had identified residues on the surface of FabF and FabI as being critical to the EcACP interaction with each enzyme, and each of these residues were systematically mutated to either alanine or a charge swap mutation. The effect of these mutations on the PPIs between EcACP and FabF and EcACP and FabI was studied using an adapted 4-DMN-EcACP assay. It was found that some point mutations could effectively destroy the PPI, while others left the PPI relatively unchanged. This work is significant in future PPI engineering efforts in being able to rapidly identify which residues on the surface of EcACP partner enzymes are most critical to the PPI. Finally, the same 4-DMN-pantetheine fluorescent tool was used to label individual bacterium in vivo from the microbiome of a tunicate. Sorting of these fluorescently labeled bacteria, followed by single-cell genomics and genome mining revealed that bacteria that up took the 4-DMN-pantetheine dye had a larger number of active secondary metabolite pathways compared to untreated samples. This study provides a workflow for the future discovery of secondary metabolite pathways from uncultured bacteria that might otherwise be overlooked by more traditional genome mining methods.

Discovery of drugs rapidly and effectively is an important aspect for Alzheimer's disease (AD). In this study, a novel high-throughput screening (HTS) method aims at screening the small-molecules with amyloid-β (Aβ) binding affinity from natural medicines, based on the combinational use of biolayer interferometry (BLI) and ultra-high-performance liquid chromatography coupled with diode-array detector and quadrupole/time-of-flight tandem mass spectrometry (UHPLC−DAD-Q/TOF-MS/MS) has been firstly developed. Briefly, the components in natural medicines disassociated from biotinylated Aβ were collected to analyze their potential Aβ binding affinity by UHPLC−DAD-Q/TOF-MS/MS. Here, baicalein was confirmed to exhibit the highest binding affinity with Aβ in Scutellaria baicalensis. Moreover, polyporenic acid C (PPAC), dehydrotumulosic acid (DTA), and tumulosic acid (TA) in Kai-Xin-San (KXS) were also identified as potent Aβ inhibitors. Further bioactivity validations indicated that these compounds could inhibit Aβ fibrillation, improve the viability in Aβ-induced PC-12 cells, and decrease the Aβ content and improve the behavioral ability in Caenorhabditis elegans. The molecular docking results confirmed that PPAC, DTA, and TA possessed good binding properties with Aβ. Collectively, the present study has provided a novel and effective HTS method for the identification of natural inhibitors on Aβ fibrillation, which may accelerate the process on anti-AD drugs discovery and development.

Background: Salviae Miltiorrhizae Radix et Rhizoma (Red Sage root) is widely used in traditional Chinese medicine (TCM) for the treatment of Alzheimer’s disease (AD) with demonstrated curative effects, based on the concept of "one drug with multiple therapeutic targets," which appears to be a good strategy for AD treatment. Objective: This study aimed to develop of high-throughput screening (HTS) method for multitherapeutic target components found in complex TCMs, which are active against AD, using Red Sage root as the case study. Method: Acetylcholinesterase (AChE) inhibitors (AChEIs) from Red Sage root extracts were pre-screened by ultrafiltration-HPLC (UF-HPLC) analysis, in which AChE was added to the extract and then ultrafiltered to remove non-binding compounds. Potential AChEIs were identified by HPLC analysis of compounds bound to AChE. A microplate-based HTS was then used to quantify the AChE inhibitory activity and antioxidant activity of the pre-screened compounds. Results: Pre-screening found ten potential inhibitors, which were identified by ESI-TOF/MS; six of these were purified by semi-preparative HPLC: Oleoyl neocryptotanshinone (1), Dihydrotanshinone Ⅰ (2), Cryptotanshinone (3), Tanshinone Ⅰ (4), Tanshinone ⅡA (5) and Miltirone (6). All six compounds had good AChE inhibitory activity and weak DPPH scavenging capacity. Conclusion: This study provides a platform and technology support for the rapid discovery of multi-target components, potentially active against AD, from complex TCMs and with strong potential for adaptation to the discovery of treatments for other diseases.

Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter Rastrum with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3β phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.

In humans, there are two forms of glutaminase (GLS), designated GLS1 and GLS2. These enzymes catalyse the conversion of glutamine to glutamate. GLS1 exists as two isozymes: kidney glutaminase (KGA) and glutaminase C (GAC). Several GLS inhibitors have been identified, of which DON (6‐diazo‐5‐oxonorleucine), BPTES (bis‐2‐(5‐phenylacetamido‐1, 3, 4‐thiadiazol‐2‐yl) ethyl sulphide), 968 (5‐(3‐Bromo‐4‐(dimethylamino)phenyl)‐2,2‐dimethyl‐2,3,5,6‐tetrahydrobenzo[a]phenanthridin‐4(1H)‐one) and CB839 (Telaglenastat) are the most widely used. However, these inhibitors have variable efficacy, specificity and bioavailability in research and clinical settings, implying the need for novel and improved GLS inhibitors. Based on this need, a diverse library of 28,000 compounds from Enamine was screened for inhibition of recombinant, purified GAC. From this library, one inhibitor designated compound 19 (C19) was identified with kinetic features revealing allosteric inhibition of GAC in the µm range. Moreover, C19 inhibits anti‐CD3/CD28‐induced CD4+ T‐cell proliferation and cytokine production with similar or greater potency as compared to BPTES. Taken together, our data suggest that C19 has the potential to modulate GLS1 activity and alter metabolic activity of T cells., Discovery and characterization of the novel glutamine (GLS) inhibitor C19 by high‐throughput screening from Enamine. C19 inhibits activity of GLS1 and reduces proliferation and cytokine secretion from activated CD4+ T cells.

Suppression of cellular O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) 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-GlcNAcylation in HEK293T 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 O-GlcNAcylation changes. The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines. Using the fluorescent reporter, we screened 120 natural products and obtained one compound, sesamin, which could markedly inhibit protein O-GlcNAcylation in HeLa 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., Graphical abstract Image 1, Highlights • The reporter developed in this study is living cell-based with convenient utility. • The method can be used for high-throughput screening. • The reporter is versatile with potential applicability in the discovery of OGT/GFAT inhibitors and antitumor drugs.

Events including antibody‒antigen affinity, internalization, trafficking and lysosomal proteolysis combinatorially determine the efficiency of antibody–drug conjugate (ADC) catabolism and hence the toxicity. Nevertheless, an approach that conveniently identifies proteins requisite for payload release and the ensuing toxicity for mechanistic studies and quality assessment is lacking. Considering the plethora of ADC candidates under development, we developed a target-responsive subcellular catabolism (TARSC) approach that examines ADC catabolism and probes changes in response to targeted interferences of proteins of interest. We firstly applied TARSC to study the commercial T-DM1 and the biosimilar. We recorded unequivocal catabolic behaviors regardless of the absence and presence of the targeted interferences. Their negligible differences in TARSC profiles agreed with their undifferentiated anti-tumoral efficacy according to further in vitro viability and in vivo tumor growth assays, highlighting TARSC analysis as a useful tool for biosimilarity assessment and functional dissection of proteins requisite for ADC catabolism. Additionally, we employed TARSC to investigate the catabolic behavior of a new trastuzumab–toxin conjugate. Collectively, TARSC can not only characterize ADC catabolism at (sub)cellular level but also comprehensively determine which protein targets affect payload release and therapeutic outcomes. Future use of TARSC is thus anticipated in early-stage screening, quality assessment and mechanistic investigations of ADCs., Graphical abstract The TARSC approach, characterizing ADC catabolism at (sub)cellular level and comprehensively determining which protein targets affect payload release and hence the therapeutic outcomes, is anticipated to support ADC development.Image 1

Restoring lost heart muscle is an attractive goal for cardiovascular regenerative medicine. One appealing strategy is the therapeutic stimulation of cardiomyocyte proliferation, which inter alia remains challenging due to available assay technologies capturing the complex biology. Here, a high-throughput-formatted phenotypic assay platform was established using rodent whole heart-derived cells to preserve the cellular environment of cardiomyocytes. Several readouts allowed the quantification of cycling cardiomyocytes, including a transgenic H2B-mCherry system for unequivocal, automated detection of cardiomyocyte nuclei. A chemical genetics approach revealed pronounced species differences and furnished pan-kinase inhibitors 5 and 36 as potent and robust inducers of endoreplication and acytokinetic mitosis. Combined profiling of the commonly used p38 MAPK inhibitors SB203580 (1), SB239063 (2) and a novel set of skepinone-L (6) derivatives pointed to off-target effects beyond p38 that might be critical for effective cardiomyocyte cytokinesis. Kinome-focused screening eventually furnished TG003 (38) as a novel candidate for stimulating cardiomyocyte proliferation.

Adenosine deaminases acting on RNA 1 (ADAR1) are enzymes involved in editing adenosine to inosine in the dsRNAs of cells associated with cancer development. The p150 isoform of ADAR1 is the only isoform containing the Zα domain that binds to both Z-DNA and Z-RNA. The Zα domain is suggested to modulate the immune response and could be a suitable target for antiviral treatment and cancer immunotherapy. In this study, we aimed to identify potential inhibitors for ADAR1 protein that bind the Zα domain using molecular docking and simulation tools. Virtual docking and molecular dynamics simulation approaches were used to screen the potential activity of 2115 FDA-approved compounds on the Zα domain of ADAR1 and filtered for to obtain the top-scoring hits. The top three compounds with the best XP Gscore—namely alendronate (−7.045), etidronate (−6.923), and zoledronate (−6.77)—were subjected to 50 ns simulations to characterize complex stability and identify the fundamental interactions that contribute to inhibition of the ADAR1 Zα domain. The three compounds were shown to interact with Lys169, Lys170, Asn173, and Tyr177 of the Zα domain-like helical backbone of Z-RNA. The study provides a comprehensive and novel insights of repurposes drugs for the inhibition of ADAR1 function.

Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.

Understanding and broad screening Li interaction energetics with surfaces are key to the development of materials for a wide range of applications including Li-based electrochemical capacitors, Li sensors, Li separation membranes, and Li-ion batteries. In this work, we build a high-throughput screening scheme to screen Li adsorption energetics on 2D metallic materials. First, density functional theory and graph convolution networks are utilized to calculate the minimum Li adsorption energies for some 2D metallic materials. The data is then used to find a dependence of the minimum Li adsorption energies on the sum of ionization potential, work function of the 2D metal, and coupling energy between Li+ and substrate, and the dependence is used to screen all 2D metallic materials. Physics-simplified learning by splitting the property into different contributions and learning or calculating each component is shown to have higher accuracy and transferability for machine learning of complex materials properties.

There has been an increase in the reported cases of tuberculosis, a disease caused by Mycobacterium tuberculosis, which is still currently affecting most of the world’s population, especially in resource-limited countries. The search for novel antitubercular chemotherapeutics from underexplored natural sources is therefore of paramount importance. The renewed interest in studies related to natural products, driven partly by the growing incidence of MDR-TB, has increased the prospects of discovering new antitubercular drug leads. This is because most of the currently available chemotherapeutics such as rifampicin and capreomycin used in the treatment of TB were derived from natural products, which are proven to be an abundant source of novel drugs used to treat many diseases. To meet the global need for novel antibiotics from natural sources, various strategies for high-throughput screening have been designed and implemented. This review highlights the current antitubercular drug discovery strategies from natural sources.

Regulators of G protein signaling (RGS) proteins serve as critical regulatory nodes to limit the lifetime and extent of signaling via G protein-coupled receptors (GPCRs). Previously, approaches to pharmacologically inhibit RGS activity have mostly focused on the inhibition of GTPase activity by interrupting the interaction of RGS proteins with the G proteins they regulate. However, several RGS proteins are also regulated by association with binding partners. A notable example is the mammalian RGS7 protein, which has prominent roles in metabolic control, vision, reward, and actions of opioid analgesics. In vivo, RGS7 exists in complex with the binding partners type 5 G protein β subunit (Gβ5) and R7 binding protein (R7BP), which control its stability and activity, respectively. Targeting the whole RGS7/Gβ5/R7BP protein complex affords the opportunity to allosterically tune opioid receptor signaling following opioid engagement while potentially bypassing undesirable side effects. Hence, we implemented a novel strategy to pharmacologically target the interaction between RGS7/Gβ5 and R7BP. To do so, we searched for protein complex inhibitors using a time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay that measures compound-mediated alterations in the FRET signal between RGS7/Gβ5 and R7BP. We performed two HTS campaigns, each screening ~100,000 compounds from the Scripps Drug Discovery Library (SDDL). Each screen yielded more than 100 inhibitors, which will be described herein.

Recent efforts on the characterization of long non-coding RNAs (lncRNAs) revealed their functional roles in modulating diverse cellular processes. These include pluripotency maintenance, lineage commitment, carcinogenesis, and pathogenesis of various diseases. By interacting with DNA, RNA and protein, lncRNAs mediate multifaceted mechanisms to regulate transcription, RNA processing, RNA interference and translation. Of more than 173000 discovered lncRNAs, the majority remain functionally unknown. The cell type-specific expression and localization of the lncRNA also suggest potential distinct functions of lncRNAs across different cell types. This highlights the niche of identifying functional lncRNAs in different biological processes and diseases through high-throughput (HTP) screening. This review summarizes the current work performed and perspectives on HTP screening of functional lncRNAs where different technologies, platforms, cellular responses and the downstream analyses are discussed. We hope to provide a better picture in applying different technologies to facilitate functional annotation of lncRNA efficiently.

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic’s Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618., PKU patients have elevated phenylalanine levels which can result in neurological impairment. Here the authors utilize biosensor-based ultra-high-throughput screening to optimize PAL activity in a synthetic biotic platform for improved in vivo performance.

Electrophoretic mobility shift assay (EMSA) technology has been widely employed for the analysis of transcription factors such as Forkhead box protein M1 (FOXM1). However, the application of high-throughput screening (HTS) in performing, such analyses are limited as it uses time consuming electrophoresis procedure and radioisotopes. In this study, we developed a FOXM1-DNA binding domain (DBD) binding assay based on time-resolved fluorescence energy transfer (TR-FRET) that enables HTS for the inhibitors of FOXM1-DNA interaction. This assay was robust, highly reproducible and could be easily miniaturized into 384-well plate format. The signal-to-background (S/B) ratio and Z' factor were calculated as 7.46 and 0.74, respectively, via a series of optimization of the assay conditions. A pilot library screening of 1019 natural compounds was performed using the FOXM1-DBD binding assay. Five hit compounds, namely, AC1LXM, BRN5, gangaleoidin, leoidin, and roemerine were identified as the inhibitors of FOXM1. In a cell viability assay, it was demonstrated that cell proliferation of FOXM1 overexpressed cell lines was suppressed in cell lines such as MDA-MB-231 and MCF-7 by five hit compounds. These results indicate that developed FOXM1-DBD binding assay can be applied to highly efficiency HTS of compound libraries.

d-Allulose is an attractive noncaloric sugar substitute with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability under harsh industrial reaction conditions hampered its practical applications. Here, we developed a continuous spectrophotometric assay (CSA) for the efficient analysis of d-allulose in a mixture. Furthermore, a high-throughput screening strategy for DAEases was developed using CSA by coupling DAEase with a NADH-dependent ribitol dehydrogenase, enabling high-throughput screening of DAEase variants with desired properties. The variant M15S/P40N/S209N exhibited a half-life of 22 h at 60 °C and an 8.7 °C increase of the T5060 value, with a 1.2-fold increase of activity. Structural modeling and molecular dynamics simulations indicated that the improvement of thermostability and activity was due to some new hydrogen bonds between chains at the dimer interface and between the residue and the substrate d-fructose. This work offers a robust tool and theoretical basis for the improvement of DAEases, which will benefit the enzymatic biosynthesis of d-allulose and promote its industrial application.

Canavan disease (CD) is a progressive, fatal neurological disorder that begins in infancy resulting from a mutation in aspartoacyclase (ASPA), an enzyme that catalyzes the deacetylation of N-acetyl aspartate (NAA) into acetate and aspartate. Increased NAA levels in the brains of affected children are one of the hallmarks of CD. Interestingly, genetic deletion of N-acetyltransferase-8-like (NAT8L), which encodes aspartate N-aceyltransferase (ANAT), an enzyme responsible for the synthesis of NAA from l-aspartate and acetyl-CoA, leads to normalization of NAA levels and improvement of symptoms in several genetically engineered mouse models of CD. Therefore, pharmacological inhibition of ANAT presents a promising therapeutic strategy for treating CD. Currently, however, there are no clinically viable ANAT inhibitors. Herein we describe the development of fluorescence-based high throughput screening (HTS) and radioactive-based orthogonal assays using recombinant human ANAT expressed in E. coli. In the fluorescence-based assay, ANAT activity was linear with respect to time of incubation up to 30 min and protein concentration up to 97.5 ng/μL with Km values for l-aspartate and acetyl-CoA of 237 μM and 11 μM, respectively. Using this optimized assay, we conducted a pilot screening of a 10 000-compound library. Hits from the fluorescence-based assay were subjected to an orthogonal radioactive-based assay using L-[U-14C] aspartate as a substrate. Two compounds were confirmed to have dose-dependent inhibition in both assays. Inhibitory kinetics studies of the most potent compound revealed an uncompetitive inhibitory mechanism with respect to l-aspartate and a noncompetitive inhibitory mechanism against acetyl-CoA. The screening cascade developed herein will enable large-scale compound library screening to identify novel ANAT inhibitors as leads for further medicinal chemistry optimization.

Peptides are small molecules composed of amino acids linked together by peptide bonds. The targeted action of these peptides along with their magnificent ability to reach locations in the body that are complicated to access, is being considered of tremendous potential in disease modifying therapies. Synthetic as well as natural peptides like Carnosine are currently under research for the treatment of Neurodegenerative Disorders (NDDs). Peptide based vaccines are currently under immense research for diseases like dementia. Toxicity of peptide-based drugs towards eukaryotic cells due to their increased haemolytic activity is of major concern and this is being tackled by introducing modifications into the peptide structure. Some crucial peptide inhibitors currently in use for neurodegenerative disorders include Aβ (16-20) KLVFF for Alzheimer’s disease, NAPVSIPQ (NAP) for Parkinson’s disease, towards eukaryotic cells Vasoactive Intestinal Peptides (VIP) for Huntington’s disease, Polyglutamine Binding Peptide-1(QBP1) for Dentatorubral- pallidoluysian atrophy (DRPLA). Certain peptides are involved in inhibition of Mitochondrial Permeability Transition (MPT) that plays a prominent part in the materialization of neurodegenerative diseases, one such example of peptides being Ba-V which is obtained from Bothrops atrox snake venom. New therapeutic peptides are being identified using bioinformatics tools like High Throughput Screening (HTS). These tools are being used to explore the selectivity, stability, extent of immune response and toxic side effects of peptides. Apart from neurodegenerative diseases, the potential of bioactive peptides is also being tested against cancer, diabetes and microbes. This review focuses on the recent advances in peptide therapeutics and novel peptides discovered for the treatment of NDs.

Interactions between membrane proteins within a cellular environment are crucial for all living cells. Robust methods to screen and analyse membrane protein complexes are essential to shed light on the molecular mechanism of membrane protein interactions. Most methods for detecting protein:protein interactions (PPIs) have been developed to target the interactions of soluble proteins. Bimolecular fluorescence complementation (BiFC) assays allow the formation of complexes involving PPI partners to be visualized in vivo, irrespective of whether or not these interactions are between soluble or membrane proteins. In this study, we report the development of a screening approach which utilizes BiFC and applies flow cytometry to characterize membrane protein interaction partners in the host Saccharomyces cerevisiae. These data allow constructive complexes to be discriminated with statistical confidence from random interactions and potentially allows an efficient screen for PPIs in vivo within a high-throughput setup.