High-throughput Library Screening Research Articles

Yeast Display for the Identification of Peptide-MHC Ligands of Immune Receptors.

T cells detect peptide antigens presented by major histocompatibility complex (MHC) proteins via their T cell receptor (TCR). The sequence diversity of possible antigens, with trillions of potential peptide-MHC targets, makes it challenging to study, characterize, and manipulate the peptide repertoire of a given TCR. Yeast display has been utilized to study the interactions between peptide-MHCs and T cell receptors to facilitate high-throughput screening of peptide-MHC libraries. Here we present insights on designing and validating a peptide-MHC yeast display construct, designing and constructing peptide libraries, conducting selections, and preparing, processing, and analyzing peptide library sequencing data. Applications for this approach are broad, including characterizing peptide-MHC recognition profiles for a TCR, screening for high-affinity mimotopes of known TCR-binding peptides, and identifying natural ligands of TCRs from expanded T cells.

A Screening Method for P450 BM3 Mutant Libraries Using Multiplexed Capillary Electrophoresis for Detection of Enzymatically Converted Compounds.

Capillary electrophoresis (CE) is an analytical method in which charged species are separated by attraction or repulsion performed in submillimeter diameter capillaries or micro- and nanofluidic channels through the application of a high voltage electric field. When capillary electrophoresis is assembled in a multicapillary instrument such as 96-well format (multiplexed), it becomes a powerful high-throughput system with the ability to simultaneously screen several types of samples like genetic mutations, metabolomes, kinase inhibitors, or enzymatic activities to name a few. The usage of a 96-multiplexed capillary electrophoresis system (96-MP-CE) represents a new platform for product-specific high-throughput screening of enzyme mutant libraries from directed evolution campaigns providing a comprehensive view on enzyme activity through the detection of all products formed. We describe the application of 96-MP-CE to screen mutant libraries of P450 BM3. MP-CE was used in directed evolution campaigns toward benzo-1,4-dioxane and α-isophorone.

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL−1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL−1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL−1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

Chemoinformatic Characterization of Synthetic Screening Libraries Focused on Epigenetic Targets.

The importance of epigenetic drug and probe discovery is on the rise. This is not only paramount to identify and develop therapeutic treatments associated with epigenetic processes but also to understand the underlying epigenetic mechanisms involved in biological processes. To this end, chemical vendors have been developing synthetic compound libraries focused on epigenetic targets to increase the probabilities of identifying promising starting points for drug or probe candidates. However, the chemical contents of these data sets, the distribution of their physicochemical properties, and diversity remain unknown. To fill this gap and make this information available to the scientific community, we report a comprehensive analysis of eleven libraries focused on epigenetic targets containing more than 50,000 compounds. We used well-validated chemoinformatics approaches to characterize these sets, including novel methods such as automated detection of analog series and visual representations of the chemical space based on Constellation Plots and Chemical Library Networks. This work will guide the efforts of experimental groups working on high-throughput and medium-throughput screening of epigenetic-focused libraries. The outcome of this work can also be used as a reference to design and describe novel focused epigenetic libraries.

Dysidenin from the Marine Sponge Citronia sp. Affects the Motility and Morphology of Haemonchus contortus Larvae In Vitro.

High-throughput screening of the NatureBank marine extract library (n = 7616) using a phenotypic assay for the parasitic nematode Haemonchus contortus identified an active extract derived from the Australian marine sponge Citronia sp. Bioassay-guided fractionation of the CH2Cl2/MeOH extract from Citronia sp. resulted in the purification of two known hexachlorinated peptides, dysidenin (1) and dysideathiazole (2). Compound 1 inhibited the growth/development of H. contortus larvae and induced multiple phenotypic changes, including a lethal evisceration (Evi) phenotype and/or somatic cell and tissue destruction. This is the first report of anthelmintic activity for these rare and unique polychlorinated peptides.

Exploring potential of Plasmodium RUVBL proteins as anti-malarial drug target

Although malaria related cases and deaths have consistently declined over time, growing resistance to existing anti-malarial drugs in Plasmodium remains a matter of extreme concern. Since we rely so heavily on use of chemotherapy for malaria treatment and knowing that all the available anti-malarial drug will become virtually useless in the near future, we have to increase our understanding of basic biology of the parasite as well as characterize new molecular targets that can be exploited for anti-malarial therapy. In the present study, PfRUVBLs (AAA family member proteins) were evaluated for their potential as novel anti-malarial drug target candidates, using computational approaches. Virtual High-throughput screening of various pharmacophore libraries obtained from three different databases (which included, Asinex, ZINC15 & PubChem) followed by extra precision docking, resulted in identification of relevant hit compounds that showed binding affinity with the active region of PfRUVBL1 protein. Based on molecular docking data, MD simulations, and protein-ligand interaction studies, combined with toxicity assessment & ADME profiling data, at least three best hits were eventually identified that could be novel potent inhibitors of PfRUVBL1 protein and can be further tested for anti-malarial activity using in vitro protocols. Communicated by Ramaswamy H. Sarma

Lamprey Variable Lymphocyte Receptor Monoclonal Antibodies for Whole-Cell Surface Antigens.

Lamprey antibodies, the variable lymphocyte receptor B proteins (VLRB), have unique properties that make them promising alternatives to jawed vertebrate immunoglobulin domain antibodies. These leucine-rich repeat proteins exhibit a diversity on par with that of jawed vertebrate antibodies but are structurally completely distinct. VLRB antibodies have been successfully raised to a variety of antigens. A procedure for high-throughput screening of full-length lamprey VLRB libraries using whole cells is described here. Lamprey antibodies against cell surface antigens can be generated and screened quickly using this method.

Rational Combination of CRM1 Inhibitor Selinexor and Olaparib Shows Synergy in Ovarian Cancer Cell Lines and Mouse Models.

CRM1 inhibitors have demonstrated antitumor effects in ovarian and other cancers; however, rational combinations are largely unexplored. We performed a high-throughput drug library screen to identify drugs that might combine well with selinexor in ovarian cancer. Next, we tested the combination of selinexor with the top hit from the drug screen in vitro and in vivo Finally, we assessed for mechanisms underlying the identified synergy using reverse phase protein arrays (RPPA). The drug library screen assessing 688 drugs identified olaparib (a PARP inhibitor) as the most synergistic combination with selinexor. Synergy was further demonstrated by MTT assays. In the A2780luc ip1 mouse model, the combination of selinexor and olaparib yielded significantly lower tumor weight and fewer tumor nodules compared with the control group (P < 0.04 and P < 0.03). In the OVCAR5 mouse model, the combination yielded significantly fewer nodules (P = 0.006) and markedly lower tumor weight compared with the control group (P = 0.059). RPPA analysis indicated decreased expression of DNA damage repair proteins and increased expression of tumor suppressor proteins in the combination treatment group. Collectively, our preclinical findings indicate that combination with selinexor to expand the utility and efficacy of PARP inhibitors in ovarian cancer warrants further exploration.

Screening of a Small Molecule Compound Library Identifies Toosendanin as an Inhibitor Against Bunyavirus and SARS-CoV-2.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus causing serious infectious disease with a high case-fatality of up to 50% in severe cases. Currently, no effective drug has been approved for the treatment of SFTSV infection. Here, we performed a high-throughput screening of a natural extracts library for compounds with activities against SFTSV infection. Three hit compounds, notoginsenoside Ft1, punicalin, and toosendanin were identified for displaying high anti-SFTSV efficacy, in which, toosendanin showed the highest inhibition potency. Mechanistic investigation indicated that toosendanin inhibited SFTSV infection at the step of virus internalization. The anti-viral effect of toosendanin against SFTSV was further verified in mouse infection models, and the treatment with toosendanin significantly reduced viral load and histopathological changes in vivo. The antiviral activity of toosendanin was further expanded to another bunyavirus and the emerging SARS-CoV-2. This study revealed a broad anti-viral effect of toosendanin and indicated its potential to be developed as an anti-viral drug for clinical use.

Manipulating MEF2C: Discovering Novel Drugs to Target ETP-ALL

Ectopic MEF2C expression due to rearrangements have been recurrently found in patients with human early thymocyte progenitor acute lymphoblastic leukemia (ETP-ALL). ETP-ALL is the most immature subtype of T-ALL and is characterized by the expression of myeloid lineage markers and an ETP-like gene expression profile. In normal haematopoiesis, MEF2C expression occurs at immature stages and in myeloid- and B cell lineages but is absent in early T cell progenitors.Here, we investigate MEF2C as an oncogene for ETP-ALL. First, we validated our initial observations in an independent T-ALL cohort that ETP-ALL patients highly express MEF2C along with BCL2, HHEX, LMO2 and LYL1 in contrast to other T-ALL subtypes. In addition, we have demonstrated that enforced MEF2C expression in the T-lineage drives a bi-phenotypic CD3 +CD19 + leukemia in mice by inhibiting a T-cell developmental program in favor a B-cell transcriptional program and that resembles human ETP-ALL. This confirms MEF2C as an oncogenic driver of ETP-ALL.We developed a model system that enabled screening of various drug libraries for compounds that overcome developmental arrest induced by MEF2C-expression, as evidenced by the upregulation of CD3 and TCRγδ. The ETP-ALL-like LOUCY cells express MEF2C as consequence of a del5(q14-qter). Further induction of MEF2C levels (i.e. LOUCY-iMEF2C cells) blocked differentiation of LOUCY cells towards a CD3 +/TCRγδ + phenotype on OP9-DL1 or DLL4-coated plates in contrast to parental LOUCY cells. This model enabled high-throughput screening of drug libraries using a flow cytometry-based assay for drugs that further promote differentiation of LOUCY-MEF2C cells. We identified various epigenetic inhibitors that promoted differentiation of LOUCY-iMEF2C cells. Unlike AML patients, where HDAC4 was shown to play an important role in the activation of MEF2C and the propagation of the leukemia, we found that different HDACs are involved that can block or promote MEF2C function. This suggests that the role of different HDACs in MEF2C regulation is cell-type or disease dependent.In addition, over-expression of MEF2C raised cellular resistance towards prednisolone treatment. Conversely, inhibition of MEF2C sensitized cells to prednisolone cytotoxicity, even in the presence of MEF2C-overexpression. Together, these data show that MEF2C is an oncogenic driver of ETP-ALL, and ectopic expression of MEF2C negatively affects steroid sensitivity. Specific targeting of only those HDACs that promote MEF2C activity may provide new therapeutic options in ETP-ALL by inhibiting MEF2C function and enhancing steroid sensitivity. DisclosuresNo relevant conflicts of interest to declare.

Assembly of Long-Adapter Single-Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets.

Genome DNA sequencing has become an affordable means to resolve questions about the genetic background of life. However, the biological functions of many DNA-encoded sequences are still relatively unknown. A highly scalable and cost-effective cloning method to select natural DNA targets from genomic templates is therefore urgently needed to enable rapid understanding of the biological products of genomes. One such method involves LASSO probes, which are long single-stranded DNA oligonucleotides designed with a universal adapter that is used to link two sequences that are complementary to a genomic target of interest. Through a pooled assembly method, LASSOs can be made for multiplex DNA capture. Herein, we describe a robust, efficient method to assemble LASSO probe libraries using a Cre-recombinase-mediated reaction and a protocol for multiplex genome target capture. The starting components are a pre-LASSO probe library comprising short DNA oligo pools designed in silico and an Escherichia coli plasmid (pLASSO) that incorporates the pre-LASSO library. Through internal recombination of pLASSO with its inserts, a mature LASSO library in final configuration can be made with high purity. Assembly of a LASSO probe library takes 4 days, and target capture can be performed in a single day. With an exponentially growing list of new genomes available for investigation, this method can enable the rapid production of ORFeome libraries for high-throughput screening to identify biological functions as a complementary approach to understand genome functional biology. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assembly of LASSO probes Support Protocol 1: Generation of pLASSO vectors Support Protocol 2: Preparation of pre-LASSOs Basic Protocol 2: Massively parallel capture of large DNAs using LASSO probes.

Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering

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.

Discovery of Antimicrobial Peptide Macrocycles Through Bacterial Display.

Peptide macrocycles exhibit great ability to inhibit bacterial growth making them a promising new avenue for antimicrobial discovery. Surface Localized Antimicrobial Display (SLAY) is a platform allowing the high-throughput screening of large peptide libraries of diverse length, composition, or structure for their antimicrobial activity, including macrocyclic peptides cyclized through disulfide bonding. Here we describe the procedure for the design and construction of a SLAY peptide library and the process for screening that library for antimicrobial potential.

Discovery and Validation of a Compound to Target Ewing's Sarcoma.

Ewing’s sarcoma, characterized by pathognomonic t (11; 22) (q24; q12) and related chromosomal ETS family translocations, is a rare aggressive cancer of bone and soft tissue. Current protocols that include cytotoxic chemotherapeutic agents effectively treat localized disease; however, these aggressive therapies may result in treatment-related morbidities including second-site cancers in survivors. Moreover, the five-year survival rate in patients with relapsed, recurrent, or metastatic disease is less than 30%, despite intensive therapy with these cytotoxic agents. By using high-throughput phenotypic screening of small molecule libraries, we identified a previously uncharacterized compound (ML111) that inhibited in vitro proliferation of six established Ewing’s sarcoma cell lines with nanomolar potency. Proteomic studies show that ML111 treatment induced prometaphase arrest followed by rapid caspase-dependent apoptotic cell death in Ewing’s sarcoma cell lines. ML111, delivered via methoxypoly(ethylene glycol)-polycaprolactone copolymer nanoparticles, induced dose-dependent inhibition of Ewing’s sarcoma tumor growth in a murine xenograft model and invoked prometaphase arrest in vivo, consistent with in vitro data. These results suggest that ML111 represents a promising new drug lead for further preclinical studies and is a potential clinical development for the treatment of Ewing’s sarcoma.

Discovery of Natural Products as Multitarget Inhibitors of Insect Chitinolytic Enzymes through High-Throughput Screening.

Small-molecule inhibitors of insect chitinolytic enzymes are potential insecticides. However, the reported inhibitors that target one enzyme usually exhibit unsatisfactory bioactivity. On the basis of the multitarget strategy, we performed a high-throughput screening of a natural product library to find insecticide leads against four chitinolytic enzymes from the Asian corn borer Ostrinia furnacalis (OfChtI, OfChtII, OfChi-h, and OfHex1). Several phytochemicals were discovered to be multitarget inhibitors of these enzymes and were predicted to occupy the -1 substrate-binding subsite and engage in polar interactions with catalytically important residues. Shikonin and wogonin, which had good inhibitory activities toward all four enzymes, also exhibited significant insecticidal activities against lepidopteran agricultural pests. This study provides the first example of using a multitarget high-throughput screening strategy to exploit natural products as insecticide leads against chitin biodegradation during insect molting.

Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells.

PurposeThe chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential.MethodsThe Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed.ResultsEx ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM.ConclusionsTaken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

Predicting base editing outcomes with an attention-based deep learning algorithm trained on high-throughput target library screens

Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable transition of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have great potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on a library of 28,294 lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.

Method for high-throughput antifungal activity screening of bacterial strain libraries

Our laboratory developed a high-throughput screen for antifungal phenotypes in which bacterial libraries in 96-well format are transferred to agar pour plates inoculated with spores of the target fungus. This method is an improvement over bacterial/fungal coculture plate methods that measure antifungal activity as inhibition of radial fungal growth.

Identification of Potential Probiotics Producing Bacteriocins Active against Listeria monocytogenes by a Combination of Screening Tools.

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow’s milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis.

High-Throughput Enzyme Assay for Screening Inhibitors of the ZDHHC3/7/20 Acyltransferases.

As enzymes that mediate the attachment of long-chain fatty acids to cysteine residues, ZDHHC proteins have been reported to be promising therapeutic targets for treating cancer and autoimmune diseases. Yet, due to the lack of potent selective inhibitors, scrutiny of the biological functions of ZDHHCs has been limited. The main hindrance for developing ZDHHC inhibitors is the lack of a facile high-throughput assay. Here, we developed a ZDHHC3/7/20 high-throughput assay based on the acylation-coupled lipophilic induction of polarization (Acyl-cLIP) method and screened several potential ZDHHC inhibitors. Furthermore, we demonstrated that in vitro results from the Acyl-cLIP assay are supported by the results from cell-based assays. We envision that this new ZDHHC3/7/20 Acyl-cLIP assay will accelerate the high-throughput screening of large compound libraries for improved ZDHHC inhibitors and provide therapeutic benefits for cancer and autoimmune diseases.