High Throughput Screening

HTS is a high-tech way to hasten the drug discovery process, allowing quick and efficient screening of large compound libraries at a rate of a few thousand compounds per day or per week.

From: Encyclopedia of Bioinformatics and Computational Biology, 2019

Drug Discovery Technologies

Z. Liu, ... J. Zhou, in Comprehensive Medicinal Chemistry III, 2017

2.13.4.2 High-Throughput Screening

High-throughput screening (HTS) is a widely used method for discovering hits in traditional targets. When it is applied to PPIs, the first problem lies in low hit rates due to the different chemical space of PPIs from traditional compound libraries. The HTS hit rate is as low as 0.0001% in the identification of JIP-JNK inhibitors performed by Chen et al.164 Tremendous efforts have been made to build new libraries suitable for PPIs such as peptide libraries,165–167 α-helix mimetic library,168 iPPI-DB,169 and natural product libraries. The most widely used HTS techniques for PPIs include the two-hybrid assay, affinity purification, fluorescence polarization (FP), and fluorescence resonance energy transfer (FRET).170–172 One of the advantages with HTS is that it works even when the structure information of a target protein is not available. In addition, HTS can also be applied to reveal inducible pockets in protein–protein interfaces, as well as allosteric modulators.16 However, the limitations include the low hit rate due to incompatible libraries and the potential false positives, which need to be eliminated. The discovery of nutlins and benzodiazepinediones as p53–MDM2 inhibitors is one of the most successful examples in identifying PPI inhibitors through HTS.173,174 RG7112, a nutlin derivative, is currently in Phase I clinical trials for the treatment of leukemia and solid tumors.175,176

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MECHANISM-BASED HIGH-THROUGHPUT SCREENING FOR NOVEL ANTICANCER DRUG DISCOVERY

Wynne Aherne, ... Paul Workman, in Anticancer Drug Development, 2002

A. Introduction

HTS plays an essential role in the drug discovery process (Fig. 1). As described above, widespread implementation of this key component has been driven by the success of genomic approaches for novel drug target identification and validation. HTS also provides the means of evaluating the large numbers of compounds available in various compound collections and those provided by combinatorial and parallel synthesis. The growth of HTS has been fueled by rapid concurrent innovations in molecular biology, assay technologies and equipment, as well as automation and information technology. Many of these developments have occurred as a result of close collaboration and partnerships between the pharmaceutical industry and various instrument and reagent manufacturers.

The drug discovery potential of HTS when coupled to a compound library with wide chemical diversity is enormous, but its success depends on several factors. These include the number and quality of validated targets, the number and diversity of compounds in the collections, and the ability to screen these in a timely and cost-effective manner using robust informative assays. Identification of a good lead using HTS can shorten drug discovery time scales considerably. However, downstream factors, such as synthetic chemistry for lead optimization and the low throughput of secondary assays for defining the pharmacological properties of active compounds, may become limiting to the overall rate of identification of candidate molecules for clinical evaluation.

If numbers of compounds screened are the only mark of success, then HTS has been enormously successful. HTS capability has increased dramatically over the last 15 years (Fig. 3). In the mid-1980s conventional bioassays, such as ligand binding assays, that require filtration or phase separation were used for screening. Using these it was possible to screen relatively small numbers of compounds. The move away from test-tube-based assays to the now almost universally used microtiter plate format marked the beginning of the growth in HTS. Over the last 10–15 years this has resulted in an almost exponential growth in screening capacity. Current estimates for throughput are in the order of 105–106 compounds per week or even in some instances per day. This phenomenal throughput is often referred to as ultra-HTS.

FIGURE 3. Approximate average screening rates during the last 15 years.

These impressive screening rates have been achieved through a professional and integrated approach to compound supply, and to developments in technologies, screening activities, automation, and data management. What is certain is that HTS (or ultra-HTS) now has the capacity to screen huge numbers of compounds. However, this capability may not run in parallel with the future needs and perceptions of the industry. The wisdom of screening enormous libraries of compounds is currently being questioned and intelligent approaches to selecting smaller subsets of compounds and the use of “focused libraries” are becoming more usual. This crucial choice is discussed later.

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Neuropeptide Receptors – Drug Development

D. Hoyer, T. Bartfai, in Encyclopedia of Neuroscience, 2009

Nonpeptide Antagonists to Neuropeptide Receptors

High-throughput screening (HTS) for neuropeptide receptor antagonists depends on our ability to engineer reporter systems using promiscuous G-proteins to couple almost any neuropeptide receptor to phospholipase C (PLC) activation and Ca2+ release from intracellular store or to the production of cyclic adenosine monophosphate (cAMP) and to cAMP responsive element-binding protein (CREB)-dependent transcription of luciferase. Such advances and the availability of additional reporter systems led to the screening of large chemical libraries for ligands to neuropeptide receptors.

Thus, nonpeptide antagonists and allosteric ligands of agonist and antagonist type have been found for many receptors that were screened, although some screens may have been unsuccessful, in spite of the large size of the libraries used. Of course, the chemical space that has been explored so far is very limited, especially given the nature of some of the targets investigated.

However, even when the HTS has identified hits, the subsequent development of drugs by pharmaceutical chemists follows the same slow and difficult path of engineering selectivity for receptor subtypes and for other relevant targets, proper safety, and determination of pharmacokinetic properties to reach the clinical candidate.

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Targeted Vectors for Cancer Gene Therapy

Jesús Gómez-NavarroDavid T. Curiel, in Encyclopedia of Cancer (Second Edition), 2002

II.D.4 Promoter Definition

High-throughput screening of phage display libraries promises to define organ- and tumor-specific ligands. In a technological breakthrough of similar potential, the rapid and precise definition of genes overexpressed and underexpressed in tumor versus normal tissues, and of the corresponding regulatory promoter sequences, is being revolutionized with other high-throughput techniques, including microarrays and serial analysis of gene expression (SAGE). For instance, based on this last method, most transcripts in tumor and normal cells can be quantitated and compared, defining gene expression profiles with a potential value that seems hard to overestimate in the current context.

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Self-emulsifying delivery systems: one step ahead in improving solubility of poorly soluble drugs

Vivek P. Chavda, Dhaval Shah, in Nanostructures for Cancer Therapy, 2017

1 Introduction

Developing the most cost-effective dosage form with enhanced efficacy of an existing drug seems to be the primary goal of pharmaceutical research that is addressed by the formulation scientist. There are numerous types of drug delivery systems that have been developed with a goal to enhance drug targeting; among them the colloidal drugs delivery system has shown great potential (Lipnski, 2000). The oral route has gained priority in the worldwide drug delivery market. Formulation scientists have always shown soft corner when it comes to oral delivery; credential goes to ease of drug administration, patient compliance with improved bioavailability. An added advantage of this delivery system is that it continues to be effective when treatment is to be continued for a long period, especially in chronic drug delivery system (Chavda, 2013; Chavda et al., 2012). Besides the factor of patient compliance, it also offers a cost-effective manufacturing process, which appeals to pharmaceutical companies. However, the potential for the development of an oral dosage form is sometimes limited for therapeutic agents having poor solubility (Lipnski, 2000). When a drug is administered by oral route, it has to follow mainly different phases, as shown in Fig. 25.1.

Figure 25.1. Different Phases Through Which Drug Travels in the Body

Approximately 30%–40% of all new chemical compounds engineered through a drug discovery program exhibit poor aqueous solubility, which presents a major challenge to modern drug delivery system. For better GI drug absorption, drug solubility and permeability are the main factors to consider (Lobenberg, 2000). To overcome such issues, formulation scientists have developed certain novel drug delivery systems (NDDS), for example, solid lipid nanoparticles (SLN), fast-dissolving tablets (FDT), etc (Chakraborty, 2009; Mittal, 2011; Tang et al., 2008). Their main goal is to achieve therapeutically effective plasma levels with oral drug delivery systems. It is believed that routes of administration that avoid first-pass metabolism may lead to better clinical safety and efficacy, predominantly due to lower doses that lead to a lower amount of potentially toxic metabolites (Amidon et al., 1995). It is clear from the previous discussion that when it comes to oral drug research, one should focus either on drug solubility and/or drug permeability across the GI track to achieve better oral bioavailability of active pharmaceutical agents.

High throughput screening (HTS) enabled screening of millions of compounds across in vitro assays characterized by rapid progress in molecular structure and genetics expression of receptors. Lipinski’s rule of five has been implemented as a qualitative tool or as a predictive model to ascertain oral absorption. This trend foretells that poor absorption or poor permeation is more likely when (Christopher, 2000; Lipnski, 2000):

1.

there are more than five H-bond donors,

2.

there are more than 10 H-bond acceptors,

3.

the molecular weight >500, and

4.

the calculated log P > 5.

Lipinski’s rule of five for the prediction of the basic property of a complex compound was still an “elusive target,” but due to implementation of Biopharmaceutical Classification System (BCS), which is based on the solubility and permeability governed by likely contributions of three major factors: dissolution, permeability, and solubility of the drug (Brahmankar and Jaiswal, 1995; Christopher, 2000; Khan et al., 2012). Table 25.1 provides the reader with a better understanding of developing such formulations for oral delivery.

Table 25.1. Application of SMEDDS in Various BCS Category Drugs (Laxmikant et al., 2014; Meghani, 2013; Mittal, 2011; Gershanik and Benita, 2000)

BCS ClassSolubility and PermeabilityRate Limiting StepApplicationProblems
BCS class IHigh solubility and high permeabilityRate of dissolution limits the in vivo absorption rateRapid dissolution of drug with improved absorption. For example, ketoprofen, tapentadol HCL, zidovudineDegradation might occur due to gut wall efflux and enzymatic action
BCS class IILow solubility and high permeabilitySolubility limits absorption fluxDissolution is a rate limiting step. For example, nisoldipine, modafinil, bosentan Monohydrate, lamivudineSolubilization and limited bioavailability
BCS class IIIHigh solubility and low permeabilityPermeability is rate determiningbioavailability may be incomplete if drug is not released and dissolved within absorption window. For example, cimetidine, ranitidine, amikacin sulfateEnzymatic degradation, gut wall efflux, and limited bioavailability
BCS class IVLow solubility and low permeabilityNo in vitro/in vivo correlation is expectedGestured difficulty to formulate hence an alternate route of administration may be needed. For example, hydrochlorothiazide, paclitaxel, furosemideSolubilization, enzymatic degradation, gut wall efflux

BCS, Biopharmaceutical Classification System; SMEDDS, self-microemulsifying drug delivery systems.

BCS class I does not have a narrow therapeutic index; that’s why it may qualify for a waiver of the very expensive BA/BE clinical testing. Such a classification can save a pharmaceutical company a considerable amount (Reddy et al., 2011).

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Screening strategies

Chayanika Putatunda, ... Abhishek Walia, in Basic Biotechniques for Bioprocess and Bioentrepreneurship, 2023

5.1 Advantages of HTS technology

HTS is well-established at the cellular and molecular levels. It combines automated and micro-quantitative experiments with analysis of large-scale data [97]. HTS was firstly developed in the early 1990s, and the subsequent increase in throughput screening further stimulated the development of technologies in assay miniaturization, automation, and robotics. HTS has now progressed into ultra-HTS screening (uHTS) [98]. HTS has now become a routine laboratory technique and is widely used in basic and applied studies in industrial microbiology and biotechnology [99]. The scope of HTS has been considerably widened with the development of new equipment such as colony pickers, liquid handling systems, fluorescence-activated cell sorting (FACS) and droplet microfluidics.

Compared with traditional screening methods, HTS has significant advantages, including: more effective automated operation—with the development of advanced equipment, automation of HTS has improved which prevents contamination and human error; fewer human resources are required—with the establishment of automated operation systems using microplates and FACS has significantly reduced the labor costs; more sensitive and accurate—new assay methods resulted in fast and accurate screening by spotting variations related to target metabolite content; lower sample volumes are required—quantification requires very small samples in microliters (in microplates) or even in nanoliters (in droplets), leading to substantial reductions in the costs of culture media and reagents.

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Imaging and Spectroscopic Analysis of Living Cells

Punita Sharma, ... Steven Finkbeiner, in Methods in Enzymology, 2012

3.1 Plate management

Plate management during HTS varies significantly with the assay and typically includes at least three key components: multiwell plate-transporting robots that move plates through the workflow, liquid-handling workstations to dispense appropriate liquids with precision, and bar-coding devices that label and track individual plates throughout the screening workflow. A wide variety of commercially available instruments for each of these tasks come with varying ranges of precision, ease-of-integration, and required user interaction to operate. A caveat of incorporating multiple instruments into a screening workflow is that a failure of one instrument can be catastrophic, as it can hold up the entire pipeline.

3.1.1 Plate-transporting robots

For HTS, robotic arms along with multiwell plate stackers automate the loading of plates precisely and continuously into our microscope. To enable round-the-clock imaging, we integrated a KiNEDx robotic arm (Peak Robotics, Colorado Springs, CO., KX-300-435-TGP), which has customized grippers that load and unload plates of transfected neurons from stackers onto the microscope stage fitted with a customized plate holder. Having a robot arm with a built-in absolute encoder is important because if an emergency stop is triggered during the screening, then the robot will be able to know its last position and resume the screening from there instead of having to restart the run from the beginning.

3.1.2 Liquid-handling workstations

Liquid-handling workstations replace manual liquid pipetting. They are timesaving, use parallel sample preparation, and provide the precision required for HTS assays. Commercially available liquid-handling systems vary in ease and extent of integration with other equipment. Some systems provide additional screen-related functionalities, such as library reformatting, cherry picking, or pin-transfer. For all our HTS-related liquid-handling tasks, we use the MicroLab Starlet workstation (Hamilton, Reno, NV.). We chose the MicroLab Starlet due to its ease of integration with a robotic arm, the large number of plate locations on the deck, and the flexible scripting language. We use an eight channel head with compressed O-ring expansion for loading and seating tips and capacitance liquid level detection for reliable pipetting. The MicroLab Starlet is also equipped with tilting capabilities to ensure complete aspiration of liquids. We developed protocols in the workstation for many screening-related tasks, from the mundane (e.g., coating and washing plates) to the arduous (e.g., lipid transfections of primary neurons).

3.1.3 Bar-coding devices

Bar coding enables the researcher to manage and track multiwell plates in HTS. Some devices offer wider functionality and flexibility by being compatible with many bar-code symbologies and consist of a reader, printer, and applicator which can read or label the plate. The plate bar code and well number enable forward and backward tracking of data from individual plates, wells, and cells. It provides exceptional security in data tracking, minimizes errors, and allows real-time data exchange. Most commercial multiwell plates for HTS come preprinted with a bar code (standard or user defined). A bar-code reader is typically incorporated at various points in a HTS system depending upon the complexity of the workflow.

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Drug Discovery Technologies

D. Ullmann, in Comprehensive Medicinal Chemistry II, 2007

3.28.6.1 Overview

Since HTS applications aim at saving precious biological material, the straightforward approach would be to reduce the assay volume. However, because in macroscopic (bulk) fluorescence techniques the signal is averaged over most of the assay volume, the signal will inevitably degrade as the volume is reduced.2 In order to overcome this limitation, read-out methods based on the detection of single fluorophores have been developed for HTS. Here, the measurement volume is microscopically small (1 fL); thus, miniaturization does not alter the measurement statistics.

The signal detected from single fluorescent molecules fluctuates due to their diffusion into and out of the excitation or detection volume, each causing a burst in fluorescence emission and, thus, in detected fluorescence photons. In SMD, these bursts from single molecules are directly analyzed. So far, SMD has generated important new results and insights into biological systems, and will play an important role in the future development of detection techniques.13,15–21,53,54 However, it needs acquisition times of at least several seconds, since a reasonable number of single-molecule events has to be gathered, in order to reach a sufficiently high accuracy. Therefore, applications for HTS purposes use other analysis methods based on the statistical analysis of the fluorescence fluctuations, which offer much lower data acquisition times. Here, more than one fluorophore can be present in the detection volume, as opposed to SMD, where at the most one fluorophore at a time is allowed.

The analysis of fluorescence signal fluctuations opens up the possibility of resolving and quantifying various components of a sample expressing different fluorescence and, hence, molecular characteristics; a feature comparable to FLA and TRA. These characteristics are directly associated with the signal fluctuations; for example, brightly fluorescing particles give rise to high fluorescence emission and detection rates and, therefore, to fluctuations with high amplitudes. Slowly diffusing fluorescing molecules remain in the detection volume and emit fluorescence over a long period of time, thus generating broader fluctuations compared with fast-diffusing fluorophores. Additionally, the fluorescence fluctuations from highly concentrated molecules show much smaller amplitudes than from molecules of low concentration. Since the fluctuating signal is influenced by a large number of molecular properties, the statistical accuracy of the characterization of a biological target will be increased by the simultaneous measurement of a variety of fluorescence parameters. Several different analysis methods have evolved over the last few years that take advantage of this molecular resolution.22–28 In contrast to SMD, these methods use the whole signal data stream to extract the information, and, therefore, the necessary data acquisition times can be lowered to below 1 s, which led to their widespread application in HTS1–9,13,15,32,44,55–57 and their integration into the FCS++ read-out portfolio of the EVOscreen HTS platform (Evotec Technologies, Hamburg, Germany). However, as outlined above, the analysis requires fluctuations of a certain amplitude, that is, the concentration of fluorescing molecules has to be close to the single-molecule level, and specialized instruments such as a confocal microscope have to be used. The different fluctuation methods are outlined further below.

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Pharmacology of G Protein Coupled Receptors

Clara C. Blad, ... Stefan Offermanns, in Advances in Pharmacology, 2011

4 Anthranilic Acid Derivatives

High-throughput screening (HTS) campaigns at a number of companies, in particular Merck, led to the discovery of anthranilic acid derivatives as HCA2 ligands, first reported by Shen et al. (2007a, 2007b). Such compounds (Schmidt et al., 2010) appear prone to have high plasma protein binding with a strong negative impact on the in vivo activity of the molecules, for example, the biphenyl compound 6 in Fig. 3. Partial hydrogenation of the anthranilic acid phenyl ring yielded compounds that retained activity on the HCA2 receptor, elaborately explored by Raghavan et al. (2008). The authors concluded that the tetrahydro variants of anthranilic acid derivatives show improved oral bioavailability and better cytochrome P450 profiles. A recent publication (Shen et al., 2010) describes the discovery of (pre)clinical candidate MK-6892 (7 in Fig. 3). It was also found (Ding et al., 2010; Schmidt et al., 2010) that the cyclohexene ring system in such compounds can be further substituted.

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Recent Advances in the Inhibition of Bacterial Fatty Acid Biosynthesis

Vincent Gerusz, in Annual Reports in Medicinal Chemistry, 2010

4.2 High-throughput screening synthetic leads

A high-throughput screening (HTS) campaign identified indolyl derivatives as S. pneumoniae FabH inhibitors. Since crystallizing this enzyme proved unsuccessful, homology modeling was used to design a more soluble analog that was cocrystallized in the E. coli homolog to facilitate rational design [23]. One of the best compounds in this series (SB418011, 14) displays IC50’s of 16 nM for S. pneumoniae, 590 nM for H. influenzae, and 1.20 µM for E. coli enzymes without any activity on the human enzyme. However, no MICs were disclosed, and the report of the S. aureus structure by the same team discusses important differences with the E. coli structure, indicating again active site architecture variability [24].

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