High-throughput Screening Technologies Research Articles

Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators.

K+ channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K+ channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K+ (Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K+ equilibrium potential (EK). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K+ (GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (KATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

Dihydroouabain, a novel radiosensitizer for cervical cancer identified by automated high-throughput screening

Background and purposeRadiotherapy plays a crucial role in the treatment of cervical cancer, but existing radiosensitizers have limited efficacy in clinical applications. The aims of this study were to establish and verify an efficient method for identifying new radiosensitizers, to use this to identify candidate radiosensitizers for cervical cancer, and to investigate the specific mechanisms of these when used in combination with radiotherapy. Materials and methodsAn automated platform for identifying radiosensitizers for cervical cancer was created based on high-throughput screening technology. The radiosensitizing effects of candidate compounds from the LOPAC1280 chemical library were evaluated in radiosensitive and radioresistant cervical cancer cells using a clonogenic survival assay, with cell cycle analyses, and western blot analyses performed for both cell lines. ResultsThe automated high-throughput screening approach identified four hit compounds. One of the most potent candidates was dihydroouabain (DHO), an inhibitor of Na+/K+-ATPase that has not previously been classified as a radiosensitizer. DHO significantly enhanced radiosensitivity in cervical cancer cells. It also abrogated radiation-induced S phase arrest in cervical cancer cells. Combination treatment significantly caused the inhibition of Chk1 and increased DNA double-strand breaks (DSB). ConclusionsDHO is a novel radiosensitizer for the treatment of cervical cancer. The automated high-throughput screening platform developed in this study proved to be powerful and effective, with the potential to be widely used in the future identification of radiosensitizers.

Rapid endothelialization of small diameter vascular grafts by a bioactive integrin-binding ligand specifically targeting endothelial progenitor cells and endothelial cells

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. Statement of SignificanceIn this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a “living” endothelium and healthy blood interface are needed.

High-Throughput Screening of Cell Transfection Enhancers Using Miniaturized Droplet Microarrays.

DNA delivery is a powerful research tool for biological research and clinical therapies. However, many nonviral transfection reagents have relatively low transfection efficiency. It is hypothesized that by treating cells with small molecules, the transfection efficiency can be improved. However, in order to identify such transfection-enhancing molecules, thousands of molecules must be tested. Current high-throughput screening (HTS) technologies based on microtiter plates are not suitable for such screenings due to the prohibitively high costs of reagents and operation. Here, the use of the droplet microarray (DMA) platform to screen 774 FDA-approved drugs with CHO-K1, Jurkat and HEK293T cells is reported. The volume of individual aqueous compartments is 20 nL, requiring 0.84 mL of cell suspension and 200 pmoles of each drug (total 0.02 moles) to perform the screening. Thus, the requirement for cells and reagents is 2500 times less than that for the same experiment performed in 384-well plates. The results reveal the potential of the DMA platform as a more cost-effective and less labor-intensive approach to HTS. Furthermore, an increase (approximately two- to fivefold) in transfection efficiency is achieved by treating cells with some molecules. This study clearly demonstrates the potential of the DMA platform for miniaturization of biochemical and cellular HTS.

Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes

The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.

A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry.

De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.

Improved method of structure-based virtual screening based on ensemble learning.

Virtual screening has become a successful alternative and complementary technique to experimental high-throughput screening technologies for drug design. Since the scoring function of docking software cannot predict binding affinity accurately, how to improve the hit rate remains a common issue in structure-based virtual screening. This paper proposed a target-specific virtual screening method based on ensemble learning named ENS-VS. In this method, protein–ligand interaction energy terms and structure vectors of the ligands were used as a combination descriptor. Support vector machine, decision tree and Fisher linear discriminant classifiers were integrated into ENS-VS for predicting the activity of the compounds. The results showed that the enrichment factor (EF) 1% of ENS-VS was 6 times higher than that of Autodock vina. Compared with the newest virtual screening method SIEVE-Score, the mean EF 1% and AUC of ENS-VS (mean EF 1% = 52.77, AUC = 0.982) were statistically significantly higher than those of SIEVE-Score (mean EF 1% = 42.64, AUC = 0.912) on DUD-E datasets; and the mean EF 1% and AUC of ENS-VS (mean EF 1% = 29.73, AUC = 0.793) were also higher than those of SIEVE-Score (mean EF 1% = 25.56, AUC = 0.765) on eight DEKOIS datasets. ENS-VS also showed significant improvements compared with other similar research. The source code is available at https://github.com/eddyblue/ENS-VS.

Recent development of nucleic acid nanosensors to detect sequence-specific binding interactions: From metal ions, small molecules to proteins and pathogens

DNA carries important genetic instructions and plays vital roles in regulating biological activities in living cells. Proteins such as transcription factors binds to DNA to regulate the biological functions of DNA, and similarly many drug molecules also bind to DNA to modulate its functions. Due to the importance of protein-DNA and drug-DNA binding, there has been intense effort in developing novel nanosensors in the same length scale as DNA, to effectively study these binding interactions in details. In addition, aptamers can be artificially selected to detect metal ions and pathogens such as bacteria and viruses, making nucleic acid nanosensors more versatile in detecting a large variety of analytes. In this minireview, we first explained the different types and binding modes of protein-DNA and drug-DNA interactions in the biological systems, as well as aptamer-target binding. This was followed by the review of five types of nucleic acid nanosensors based on optical or electrochemical detection. The five types of nucleic acid nanosensors utilizing colorimetric, dynamic light scattering (DLS), surface-enhanced Raman spectroscopy (SERS), fluorescence and electrochemical detections have been recently developed to tackle some of the challenges in high-throughput screening technology for large scale analysis, which is especially useful for drug development and mass screening for pandemic outbreak such as SARS or COVID-19.

High-Throughput Screening of Multimetal Sulfides-Modified g-C3N4 for Degradation of Organic Contaminations Based on Ink-Jet Printing (IJP) Technology

In this study, a novel method combining microplate fluorescence imaging (FI) and high-throughput screening (HTS) technology was applied to screen and evaluate the multicomponent metal (Zn, Cd, Ni) sulfides-modified g-C3N4 with high-activity photocatalytic performance. Glass screen printing was creatively used in preparing a photocatalytic reaction microplate containing 225 independent micro-reaction chambers (μRCs) as experiment carriers. A photocatalyst chip comprising 225 ZnxCdyNi1−x−yS/g-C3N4 multicomponent photocatalysts was made via chemical ink-jet printing (IJP) technology, at last 23 high-efficiency M3S/g-C3N4were screened out from the photocatalyst chip by the optical density (OD) method.

Microfluidic Technologies for High Throughput Screening Through Sorting and On-Chip Culture of C. elegans.

The nematode Caenorhabditis elegans is a powerful model organism that has been widely used to study molecular biology, cell development, neurobiology, and aging. Despite their use for the past several decades, the conventional techniques for growth, imaging, and behavioral analysis of C. elegans can be cumbersome, and acquiring large data sets in a high-throughput manner can be challenging. Developments in microfluidic “lab-on-a-chip” technologies have improved studies of C. elegans by increasing experimental control and throughput. Microfluidic features such as on-chip control layers, immobilization channels, and chamber arrays have been incorporated to develop increasingly complex platforms that make experimental techniques more powerful. Genetic and chemical screens are performed on C. elegans to determine gene function and phenotypic outcomes of perturbations, to test the effect that chemicals have on health and behavior, and to find drug candidates. In this review, we will discuss microfluidic technologies that have been used to increase the throughput of genetic and chemical screens in C. elegans. We will discuss screens for neurobiology, aging, development, behavior, and many other biological processes. We will also discuss robotic technologies that assist in microfluidic screens, as well as alternate platforms that perform functions similar to microfluidics.

Applications of Deep-Learning in Exploiting Large-Scale and Heterogeneous Compound Data in Industrial Pharmaceutical Research.

In recent years, the development of high-throughput screening (HTS) technologies and their establishment in an industrialized environment have given scientists the possibility to test millions of molecules and profile them against a multitude of biological targets in a short period of time, generating data in a much faster pace and with a higher quality than before. Besides the structure activity data from traditional bioassays, more complex assays such as transcriptomics profiling or imaging have also been established as routine profiling experiments thanks to the advancement of Next Generation Sequencing or automated microscopy technologies. In industrial pharmaceutical research, these technologies are typically established in conjunction with automated platforms in order to enable efficient handling of screening collections of thousands to millions of compounds. To exploit the ever-growing amount of data that are generated by these approaches, computational techniques are constantly evolving. In this regard, artificial intelligence technologies such as deep learning and machine learning methods play a key role in cheminformatics and bio-image analytics fields to address activity prediction, scaffold hopping, de novo molecule design, reaction/retrosynthesis predictions, or high content screening analysis. Herein we summarize the current state of analyzing large-scale compound data in industrial pharmaceutical research and describe the impact it has had on the drug discovery process over the last two decades, with a specific focus on deep-learning technologies.

Pd-Catalyzed Cross-Coupling Reactions Promoted by Biaryl Phosphorinane Ligands

We report the use of biaryl phosphorinanes as ligands for Pd-catalyzed cross-coupling reactions. A modular synthesis was developed that employs a double conjugate addition of primary biaryl phosphines into 1,1,5,5-tetraalkyl penta-1,4-diene-3-ones. Notably, this synthesis does not require the use of copper, a known contaminant in structurally related biaryl phosphane ligands. Using the synthetic strategy described above, we synthesized a library of biaryl phosphorinanes, varying their substitution about phosphorus and the steric and electronic nature of the biaryl motif. We then benchmarked their performance as ligands in Pd-catalyzed cross coupling reactions such as aryl sulfonamidation, aryl alkoxylation, and aryl amination in the presence of soluble organic bases. In each reaction studied, many ligands outperformed biaryl phosphanes known to promote the given transformation. Detailed substrate scopes were determined using high-throughput screening technology. Several biaryl phosphorinanes and their cor...

Self-coalescing flows in microfluidics for pulse-shaped delivery of reagents.

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.

Probing Specificity of Protein-Protein Interactions with Chiral Plasmonic Nanostructures.

Protein-protein interactions (PPIs) play a pivotal role in many biological processes. Discriminating functionally important well-defined protein-protein complexes formed by specific interactions from random aggregates produced by nonspecific interactions is therefore a critical capability. While there are many techniques which enable rapid screening of binding affinities in PPIs, there is no generic spectroscopic phenomenon which provides rapid characterization of the structure of protein-protein complexes. In this study we show that chiral plasmonic fields probe the structural order and hence the level of PPI specificity in a model antibody-antigen system. Using surface-immobilized Fab' fragments of polyclonal rabbit IgG antibodies with high specificity for bovine serum albumin (BSA), we show that chiral plasmonic fields can discriminate between a structurally anisotropic ensemble of BSA-Fab' complexes and random ovalbumin (OVA)-Fab' aggregates, demonstrating their potential as the basis of a useful proteomic technology for the initial rapid high-throughput screening of PPIs.

Therapeutic strategies to target the Ebola virus life cycle.

Following the Ebola virus disease epidemic in west Africa, there has been increased awareness of the need for improved therapies for emerging diseases, including viral haemorrhagic fevers such as those caused by Ebola virus and other filoviruses. Our continually improving understanding of the virus life cycle coupled with the increased availability of 'omics' analyses and high-throughput screening technologies has enhanced our ability to identify potential viral and host factors and aspects involved in the infection process that might be intervention targets. In this Review we address compounds that have shown promise to various degrees in interfering with the filovirus life cycle, including monoclonal antibodies such as ZMapp, mAb114 and REGN-EB3 and inhibitors of viral RNA synthesis such as remdesivir and TKM-Ebola. Furthermore, we discuss the general potential of targeting aspects of the virus life cycle such as the entry process, viral RNA synthesis and gene expression, as well as morphogenesis and budding.

The Hippo-YAP pathway regulates the proliferation of alveolar epithelial progenitors after acute lung injury.

Regeneration of pulmonary epithelial cells plays an important role in the recovery of acute lung injury (ALI), which is defined by pulmonary epithelial cell death. However, the mechanism of the regenerative capacity of alveolar epithelial cells is unknown. Using a lung injury mouse model induced by hemorrhagic shock and lipopolysaccharide, a protein mass spectrometry-based high-throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells (AEC2s), we analyzed the mechanism of alveolar epithelial cells proliferation. We demonstrated that the expression of Hippo-yes-associated protein 1 (YAP1) key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI. Furthermore, the results showed that YAP1+ cells in lung tissue after ALI were mainly Sftpc lineage-labeled AEC2s. An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 small interfering RNA and Hippo inhibitor. These findings revealed that YAP functioned as a key regulator to promote AEC2s proliferation, with the Hippo signaling pathway playing a pivotal role in this process.

Exploring Proteomic Drug Targets, Therapeutic Strategies and Protein - Protein Interactions in Cancer: Mechanistic View

Protein-Protein Interactions (PPIs) drive major signalling cascades and play critical role in cell proliferation, apoptosis, angiogenesis and trafficking. Deregulated PPIs are implicated in multiple malignancies and represent the critical targets for treating cancer. Herein, we discuss the key protein-protein interacting domains implicated in cancer notably PDZ, SH2, SH3, LIM, PTB, SAM and PH. These domains are present in numerous enzymes/kinases, growth factors, transcription factors, adaptor proteins, receptors and scaffolding proteins and thus represent essential sites for targeting cancer. This review explores the candidature of various proteins involved in cellular trafficking (small GTPases, molecular motors, matrix-degrading enzymes, integrin), transcription (p53, cMyc), signalling (membrane receptor proteins), angiogenesis (VEGFs) and apoptosis (BCL-2family), which could possibly serve as targets for developing effective anti-cancer regimen. Interactions between Ras/Raf; X-linked inhibitor of apoptosis protein (XIAP)/second mitochondria-derived activator of caspases (Smac/DIABLO); Frizzled (FRZ)/Dishevelled (DVL) protein; beta-catenin/T Cell Factor (TCF) have also been studied as prospective anticancer targets. Efficacy of diverse molecules/ drugs targeting such PPIs although evaluated in various animal models/cell lines, there is an essential need for human-based clinical trials. Therapeutic strategies like the use of biologicals, high throughput screening (HTS) and fragment-based technology could play an imperative role in designing cancer therapeutics. Moreover, bioinformatic/computational strategies based on genome sequence, protein sequence/structure and domain data could serve as competent tools for predicting PPIs. Exploring hot spots in proteomic networks represents another approach for developing targetspecific therapeutics. Overall, this review lays emphasis on a productive amalgamation of proteomics, genomics, biochemistry, and molecular dynamics for successful treatment of cancer.

Automating a Magnetic 3D Spheroid Model Technology for High-Throughput Screening.

Affordable and physiologically relevant three-dimensional (3D) cell-based assays used in high-throughput screening (HTS) are on the rise in early drug discovery. These technologies have been aided by the recent adaptation of novel microplate treatments and spheroid culturing techniques. One such technology involves the use of nanoparticle (NanoShuttle-PL) labeled cells and custom magnetic drives to assist in cell aggregation to ensure rapid 3D structure formation after the cells have been dispensed into microtiter plates. Transitioning this technology from a low-throughput manual benchtop application, as previously published by our lab, into a robotically enabled format achieves orders of magnitude greater throughput but required the development of specialized support hardware. This effort included in-house development, fabrication, and testing of ancillary devices that assist robotic handing and high-precision placement of microtiter plates into an incubator embedded with magnetic drives. Utilizing a "rapid prototyping" approach facilitated by cloud-based computer-aided design software, we built the necessary components using hobby-grade 3D printers with turnaround times that rival those of traditional manufacturing/development practices at a substantially reduced cost. This approach culminated in a first-in-class HTS-compatible 3D system in which we have coupled 3D bioprinting to a fully automated HTS robotic platform utilizing our novel magnetic incubator shelf assemblies.

RNA-based diagnostic and therapeutic strategies for cardiovascular disease.

Cardiovascular diseases are the leading cause of death globally and are associated with increasing financial expenditure. With the availability of next-generation sequencing technologies since the early 2000s, non-coding RNAs such as microRNAs, long non-coding RNAs and circular RNAs have been assessed as potential therapeutic targets for numerous diseases, including cardiovascular diseases. In this Review, we summarize current approaches employed to screen for novel coding and non-coding RNA candidates with diagnostic and therapeutic potential in cardiovascular disease, including next-generation sequencing, functional high-throughput RNA screening and single-cell sequencing technologies. Furthermore, we highlight viral-based delivery tools that have been widely used to evaluate the therapeutic utility of both coding and non-coding RNAs in the context of cardiovascular disease. Finally, we discuss the potential of using oligonucleotide-based molecular products such as modified RNA, small interfering RNA and RNA mimics/inhibitors for the treatment of cardiovascular diseases. Given that many non-coding RNAs have not yet been functionally annotated, the number of potential RNA diagnostic and therapeutic targets for cardiovascular diseases will continue to expand for years to come.

Adalimumab

As a powerful tool for the discovery of peptide compounds, high-throughput library screening technology can also perform multitarget screening with high chemical diversity as a condition, which accelerates the process of exploring high-affinity peptide molecules. These techniques can be generally divided into in vitro and in vivo screening techniques, both of which play important roles in peptide drug discovery. Compared with linear peptides, cyclic peptides have been proven to have good bioavailability, membrane permeability, and stability and longer half-lives and have gradually become a topic of high interest in peptide drug research. High-throughput library screening technology plays an important role in the development of cyclic peptide drugs. Based on published studies, this paper reviews the high-throughput library screening technology used in the development of cyclic peptide drugs.